Ice inhibiting compounds and uses thereof

ABSTRACT

The present disclosure identifies caspase-1/ICE as a therapeutic target for α-synuclein associated diseases and disorders. Related methods and compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 61/410,856, filed Nov. 5, 2010, and U.S.Provisional Application Ser. No. 61/410,852, filed Nov. 5, 2010, theentire contents of each of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of α-synuclein (Spillantini et al., Proc. Natl. Acad. Sci.USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999), a140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Two dominant mutations in α-synuclein causingfamilial early onset Parkinson's disease have been described, suggestingthat Lewy bodies contribute mechanistically to the degeneration ofneurons in Parkinson's disease and related disorders (Polymeropoulos etal., Science 276:2045-2047, 1997; Kruger et al., Nature Genet.18:106-108, 1998; Zarranz et al., Ann. Neurol. 55:164-173, 2004).Triplication and duplication mutations of the α-synuclein gene have beenlinked to early-onset of Parkinson's disease (Singleton et al., Science302:841, 2003; Chartier-Harlin at al. Lancet 364:1167-1169, 2004; Ibanezet al., Lancet 364:1169-1171, 2004). In vitro studies have demonstratedthat recombinant α-synuclein can indeed form Lewy body-like fibrils(Conway et al., Nature Med. 4:1318-1320, 1998; Hashimoto et al., BrainRes. 799:301-306, 1998; Nahri et al., J. Biol. Chem. 274:9843-9846,1999). Both Parkinson's disease-linked α-synuclein mutations acceleratethis aggregation process, demonstrating that such in vitro studies mayhave relevance for Parkinson's disease pathogenesis. α-Synucleinaggregation and fibril formation fulfill the criteria of anucleation-dependent polymerization process (Wood et al., J. Biol. Chem.274:19509-19512, 1999).

SUMMARY OF THE INVENTION

The present invention encompasses the finding that caspase-1 (ICE)cleaves α-synuclein in vivo. As described herein, such cleavagegenerates α-synuclein fragments that are prone to toxic aggregateformation. The present invention pertains, among other things, toICE-dependent cleavage of α-synuclein, as well as to related methodswhich are useful for the diagnosis and/or treatment of diseases anddisorders associated with ICE-cleaved α-synuclein.

In one aspect, the invention provides methods for identifying and/orcharacterizing compounds that inhibit ICE. For example, in someembodiments, the invention provides methods comprising steps of: (1)providing a plurality of test compounds; (2) contacting test compoundsfrom the plurality with full-length α-synuclein in the presence of ICE;and (3) determining whether one or more of the test compounds inhibitsICE cleavage of the full-length α-synuclein into α-synuclein fragments.In some embodiments, α-synuclein cleavage is determined by measuringrelative levels of full-length α-synuclein and cleaved α-synuclein; insome embodiments a higher ratio of full-length α-synuclein to cleavedα-synuclein in the presence of the test compound as compared to thecontrol indicates that the test compound is an ICE inhibitor thatinhibits ICE cleavage of α-synuclein.

In some embodiments of the invention, relevant α-synuclein fragmentsinclude a fragment of about 120 amino acids in length. In someembodiments of the invention, relevant α-synuclein fragments include afragment of about 20 amino acids in length. In some embodiments of theinvention, relevant α-synuclein fragments include both a fragment ofabout 120 amino acids in length and one of about 20 amino acids inlength. In some embodiments, an α-synuclein fragment of interestcorresponds to a polypeptide resulting from cleavage of full-lengthα-synuclein fragments at a site corresponding to residue 120 of SEQ IDNO:1.

In some embodiments, the invention provides antibodies specific to oneor more particular α-synuclein fragments. In some embodiments, theinvention provides antibodies specific to one or more α-synucleinfragments generated by ICE-dependent proteolysis of α-synuclein. In someembodiments, the present invention provides antibodies that bind to afull-length α-synuclein polypeptide, but do not bind to a fragment ofthat α-synuclein polypeptide that would be generated by cleavage of thefull-length α-synuclein polypeptide by ICE. In some embodiments, thepresent invention provides α-synuclein antibodies that specifically bindto a fragment generated by ICE cleavage of a full-length α-synucleinpolypeptide, but not to the full-length α-synuclein polypeptide itself.In some embodiments, antibodies provided herein specifically bind to oneor more conformational epitopes.

The present invention provides systems, including methods, foridentifying and/or characterizing α-synuclein cleaving enzymes. Forexample, in some embodiments, the present invention provides methodscomprising steps of: (1) providing a plurality of candidate enzymes(e.g., caspase enzymes) that are candidate α-synuclein cleaving enzymes;(2) contacting candidate enzymes from the plurality with a full-lengthα-synuclein; and (3) determining whether one or more of the candidateenzymes cleaves the full-length α-synuclein into fragments. In someembodiments, the step of determining comprises determining whether oneor more of the candidate enzymes cleaves the full-length α-synucleininto fragments including one that is about 120 amino acids in length.This step may involve measuring or detecting relative amounts ofα-synuclein species (e.g., full length and/or fragments). Alternativelyor additionally, the present invention provides methods comprising stepsof (1) contacting a full-length α-synuclein polypeptide with a caspaseenzyme under conditions and for a time sufficient to permit cleavage ofthe full-length α-synuclein polypeptide into fragments. In someembodiments, at least one fragment is detected.

The present invention also provides methods directed to an ICE inhibitortherapy. In some embodiments, the invention provides methods comprisingadministering to a patient suffering from or susceptible to developing asynucleinopathy disease, disorder or condition, a composition comprisingan amount of an ICE inhibitor sufficient to inhibit cleavage ofα-synuclein by ICE.

In certain embodiments, the present invention relates to asynucleinopathy disease, disorder or condition, including but are notlimited to: Parkinson's disease (such as an autosomal-dominantParkinson's disease), dementia, or multiple system atrophy. In certainembodiments, the present invention relates to a synucleinopathy disease,disorder or condition that is characterized by the presence of Lewybodies.

DEFINITIONS

Alpha-synuclein polypeptide/α-synuclein polypeptide: The term“α-synuclein polypeptide” or “alpha-synuclein,” as used herein, refersto a polypeptide that shows a high degree of sequence identity with awild type α-synuclein polypeptide such as, for example, wild type humanα-synuclein. The wild-type, full-length form of human α-synuclein is a140 amino acid polypeptide comprising the following amino acid sequence(see, for example, Accession Number: NP_(—)000336.1):

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYVGSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQKTVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA(SEQ ID NO: 1). In some embodiments, an α-synuclein polypeptide shows atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% overall sequence identity with SEQ ID NO:1. The full-lengthα-synuclein primary structure is typically divided into three distinctdomains: Residues corresponding to residues 1-60 of SEQ ID NO:1represent an amphipathic N-terminal region dominated by four 11-residuerepeats including the consensus sequence KTKEGV (SEQ ID NO: 2). Thissequence has been reported to have a structural alpha helix propensitysimilar to apolipoproteins-binding domains; residues 61-95 correspond toa central hydrophobic region which includes the non-amyloid component(NAC) region, involved in protein aggregation; and, residues 96-140 makeup a highly acidic and proline-rich region which has no distinctstructural propensity. In some embodiments, an α-synuclein polypeptidemay include one or more point mutations as compared with SEQ ID NO:1,which are associated with a disease, disorder or condition. For example,certain monogenic point mutations, including but not limited to A30P,A53T, and E46K, have been identified as causal factors of early onsetfamilial Parkinson disease.

α-synuclein fragment: The term “α-synuclein fragment,” as used herein,refers to a polypeptide having an amino acid sequence that issubstantially identical to that of an α-synuclein polypeptide exceptthat the fragment includes less than all of the amino acid residuesfound in a full-length α-synuclein polypeptide. In some embodiments afragment lacks one or more terminal residues or sections found in afull-length α-synuclein polypeptide. In some embodiments, an α-synucleinfragment is fewer than 140, 139, 138, 137, 136, 135, 134, 133, 132, 131,130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117,116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103,102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 92, 90, 89, 88, 87, 86,85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68,67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50,49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, or 10 amino acids long. In some embodiments, an α-synucleinfragment is about 120 amino acids long. In some embodiments, anα-synuclein fragment corresponds to a cleavage product of a full-lengthα-synuclein polypeptide. In some embodiments, an α-synuclein fragmentcorresponds to a product of cleavage of a full-length α-synucleinpolypeptide at a site corresponding to approximately residue 120 of SEQID NO:1.

Biological sample: The term “biological sample,” as used herein, refersto any solid or fluid sample obtained from, excreted by, or secreted byany living organism, including single-celled micro-organisms (such asbacteria and yeasts) and multicellular organisms (such as plants andanimals, for instance a vertebrate or a mammal, and in particular ahealthy or apparently healthy human subject or a human patient affectedby a condition or disease to be diagnosed or investigated). A biologicalsample can be in any form, including a solid material such as a tissue,cells, a cell pellet, a cell extract, cell homogenates, or cellfractions; or a biopsy, or a biological fluid. The biological fluid maybe obtained from any site (e.g., blood, saliva (or a mouth washcontaining buccal cells), tears, plasma, serum, urine, bile,cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleuralfluid, or cells therefrom, aqueous or vitreous humor, or any bodilysecretion), a transudate, an exudate (e.g. fluid obtained from anabscess or any other site of infection or inflammation), or fluidobtained from a joint (e.g. a normal joint or a joint affected bydisease such as rheumatoid arthritis, osteoarthritis, gout or septicarthritis). A biological sample can be obtained from any organ or tissue(including a biopsy or autopsy specimen) or may comprise cells (whetherprimary cells or cultured cells) or medium conditioned by any cell,tissue or organ. Biological samples may also include sections of tissuessuch as frozen sections taken for histological purposes. Biologicalsamples also include mixtures of biological molecules includingproteins, lipids, carbohydrates and nucleic acids generated by partialor complete fractionation of cell or tissue homogenates. In certainembodiments, a biological sample is a blood sample containingerythrocytes. Although a sample is preferably taken from a humansubject, biological samples may be from any animal, plant, bacteria,virus, yeast, etc. The term “animal,” as used herein, refers to humansas well as non-human animals, at any stage of development, including,for example, mammals, birds, reptiles, amphibians, fish, worms andsingle cells. Cell cultures and live tissue samples are considered to bepluralities of animals. In certain exemplary embodiments, the non-humananimal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey,a dog, a cat, a sheep, cattle, a primate, or a pig). An animal may be atransgenic animal or a human clone. If desired, a biological sample maybe subjected to preliminary processing, including preliminary separationtechniques. In some embodiments, a biological sample contains or isderived from one or more cells or biopolymers. Cell-containingcompositions include, for example, mammalian blood, red cellconcentrates, platelet concentrates, leukocyte concentrates, blood cellproteins, blood plasma, platelet-rich plasma, a plasma concentrate, aprecipitate from any fractionation of the plasma, a supernatant from anyfractionation of the plasma, blood plasma protein fractions, purified orpartially purified blood proteins or other components, serum, semen,mammalian colostrum, milk, saliva, placental extracts, acryoprecipitate, a cryosupernatant, a cell lysate, mammalian cellculture or culture medium, products of fermentation, ascites fluid,proteins induced in blood cells, and products produced in cell cultureby normal or transformed cells (e.g., via recombinant DNA or monoclonalantibody technology). Biological compositions can be cell-free. Incertain embodiments, a suitable biological composition or biologicalsample is a red blood cell suspension. In some embodiments, a blood cellsuspension includes mammalian blood cells. In certain embodiments, bloodcells are obtained from a human, a non-human primate, a dog, a cat, ahorse, a cow, a goat, a sheep or a pig. In certain embodiments, a bloodcell suspension includes red blood cells and/or platelets and/orleukocytes and/or bone marrow cells.

Characteristic Sequence Element: As used herein, a “characteristicsequence element” of a protein or polypeptide is one that contains acontinuous stretch of amino acids, or a collection of continuousstretches of amino acids, that together are characteristic of a proteinor polypeptide. Each such continuous stretch generally will contain atleast two amino acids. Furthermore, those of ordinary skill in the artwill appreciate that typically at least 5, at least 10, at least 15, atleast 20 or more amino acids are required to be characteristic of aprotein. In general, a characteristic sequence element is one that, inaddition to the sequence identity specified above, shares at least onefunctional characteristic (e.g., biological activity, epitope, etc) withthe relevant intact protein. In many embodiments, a characteristicsequence element is one that is present in all members of a family ofpolypeptides, and can be used to define such members.

Combination therapy: The term “combination therapy,” as used herein,refers to those situations in which two or more different pharmaceuticalagents are administered in overlapping regimens so that the subject issimultaneously exposed to both agents.

Determine: Many methodologies described herein include a step of“determining.” Those of ordinary skill in the art, reading the presentspecification, will appreciate that such “determining” can utilize anyof a variety of techniques available to those skilled in the art,including, for example, specific techniques explicitly referred toherein. In some embodiments, a determination involves manipulation of aphysical sample. In some embodiments, a determination involvesconsideration and/or manipulation of data or information, for exampleutilizing a computer or other processing unit adapted to perform arelevant analysis. In some embodiments, a determination involvesreceiving relevant information and/or materials from a source.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regiment, which may involve one or more doses. Insome embodiments, a dosing regimen comprises a plurality of doses eachof which are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses.

Isolated: The term “isolated,” as used herein, refers to an agent orentity that has either (i) been separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature or in an experimental setting); or (ii) produced by the handof man. Isolated agents or entities may be separated from at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.

Polypeptide: A “polypeptide,” generally speaking, is a string of atleast two amino acids attached to one another by a peptide bond. In someembodiments, a polypeptide may include at least 3-5 amino acids, each ofwhich is attached to others by way of at least one peptide bond. Thoseof ordinary skill in the art will appreciate that polypeptides sometimesinclude “non-natural” amino acids or other entities that nonetheless arecapable of integrating into a polypeptide chain, optionally.

Prevention: The term “prevention,” as used herein, refers to a delay ofonset, and/or reduction in frequency and/or severity of one or moresymptoms of a particular disease, disorder or condition (e.g., infectionfor example with influenza virus). In some embodiments, prevention isassessed on a population basis such that an agent is considered to“prevent” a particular disease, disorder or condition if a statisticallysignificant decrease in the development, frequency, and/or intensity ofone or more symptoms of the disease, disorder or condition is observedin a population susceptible to the disease, disorder, or condition.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues that share one or more structural and/orfunctional characteristics. For example, as is well known by those ofordinary skill in the art, certain amino acids are typically classifiedas “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar”or “non-polar” side chains In some embodiments, substitution of oneamino acid for another of the same type is considered a “homologous”substitution. Typical amino acid categorizations are summarized below:

Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive −4.5Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polar negative−3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polarnegative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly G nonpolarneutral −0.4 Histidine His H polar positive −3.2 Isoleucine Ile Inonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2 Ambiguous Amino Acids 3-Letter 1-LetterAsparagine or aspartic acid Asx B Glutamine or glutamic acid Glx ZLeucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999;all of the foregoing of which are incorporated herein by reference. Inaddition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more of their correspondingresidues are homologous over a relevant stretch of residues. In someembodiments, the relevant stretch is a complete sequence. In someembodiments, the relevant stretch is at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 125, at least 150, at least 175, at least 200, at least 225, atleast 250, at least 275, at least 300, at least 325, at least 350, atleast 375, at least 400, at least 425, at least 450, at least 475, atleast 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guideto the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999; all of the foregoing of whichare incorporated herein by reference. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or more of their corresponding residues are identicalover a relevant stretch of residues. In some embodiments, the relevantstretch is a complete sequence. In some embodiments, the relevantstretch is at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 325, at least 350, at least 375, at least 400, atleast 425, at least 450, at least 475, at least 500 or more residues.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that elicits a desired biological or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” refers to any methodused to alleviate, delay onset, reduce severity or incidence, or yieldprophylaxis of one or more symptoms or aspects of a disease, disorder,or condition. For the purposes of the present invention, treatment canbe administered before, during, and/or after the onset of symptoms.

Unit dose: The expression “unit dose” as used herein refers to aphysically discrete unit of a pharmaceutical composition, formulated foradministration to a subject. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple doses is required, orexpected to be required, in order to achieve an intended effect. Theunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may contain a variety ofcomponents in addition to the therapeutic agent(s). For example,acceptable carriers (e.g., pharmaceutically acceptable carriers),diluents, stabilizers, buffers, preservatives, etc., may be included asdescribed infra. It will be understood, however, that the total dailyusage of a formulation of the present disclosure will often be decidedby the attending physician within the scope of sound medical judgment.In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a set of SDS-PAGE images demonstrating that purified,activated caspase-1 cleaves alpha-synuclein in vitro.

FIG. 2 provides a set of SDS-PAGE images demonstrating that caspase-1inhibitor blocks the fragmentation of alpha-synuclein.

FIG. 3 provides a set of SDS-PAGE images demonstrating that oxidativestress activates caspase-1 and generates the alpha-synuclein fragment inneuroblastoma cells.

FIG. 4 provides graphs depicting results from assays measuring caspase-1cleavage of alpha-synuclein in vitro.

FIG. 5 provides an image of a Western blot of ICE digestedalpha-synuclein. Lane 1; molecular weight marker, lane 2; αSyn alone,lane 3 to 6; αSyn plus increasing amounts of ICE (2 to 12 ug).Appearance of a smaller fragment of synuclein below 17 kD with intensityincreasing with amount of ICE indicate that ICE was generating thefragment.

FIG. 6 provides an image of a Western blot of ICE digested α-syn plusinhibitor. Lane 1; molecular weight marker, lane 2; αSyn alone, lane 3;αSyn plus ICE (10 ug), lane 4; αSyn plus ICE (10 ug) and 20 uM of NCGinhibitor from Graig Thomas at NIH. Appearance of a smaller fragment ofsynuclein below 17 kD in lane 3 indicate that ICE was generating thefragment. This fragment is demished in lane 4 containing ICE specificinhibitor, indicating that ICE specifically was generating the fragment.

FIG. 7 provides MALDI-TOF mass spec analysis of ICE digested α-syn. Thefragment generated by ICE was determined to be 13167 Da whichcorresponds to residues 1-121.

FIG. 8 provides graphs depicting M17 cell viability of M17 cells withpcDNA3 empty vector in the presence of menadione and M17 cellsoverexpressing alpha-synuclein in the presence of menadione.

FIG. 9 provides an image of a Western blot of menadione treated M17cells. Lane 1; molecular weight marker, lane 2; M17 cells treated with1% DMSO as control, lane 3; M17 cells treated with 12 uM menadione in 1%DMSO, lane 4; M17 cells treated with 14 uM menadione in 1% DMSO.Appearance of a smaller fragment of synuclein below 17 kD in lane 3 and4 indicate that menadione induced fragmentation of α-synuclein.

FIG. 10 provides a bar graph depicting the effects of caspase-1inhibition and knockdown on cell viability. Inhibition of ICE with VX765or knockdown of ICE gene with shRNA rescued M17 cells overexpressingalpha-synuclein from alpha-synuclein-induced toxicity.

FIG. 11 provides a graph showing the effect of caspace-1 cleavage ofalpha-synuclein on aggregation over the course of 70 hours.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Alpha-Synuclein

Synucleins are a family of proteins composed of α-, β-, andγ-synucleins. In neurons, the synuclein proteins are localizedpredominantly at the presynaptic sites. Among the synuclein proteins,α-Synuclein is a small lipid-binding protein involved in vesicletrafficking, whose function is poorly characterized. It is of greatinterest from a clinical perspective because α-synuclein dysfunction hasbeen implicated in the pathogenesis of several neurodegenerativedisorders, including Parkinson's disease (PD) (Ian et al., ClinicalNeurosc. Res. 1:445-455, 2001; Trojanowski and Lee, Neurotoxicology23:457-460, 2002).

α-Synuclein recombinant protein, and non-Aβ component (known as NAC),which is a 35-amino acid peptide fragment of α-synuclein, both have theability to form fibrils when incubated at 37° C., and are positive withamyloid stains such as Congo red (demonstrating a red/greenbirefringence when viewed under polarized light) and Thioflavin S(demonstrating positive fluorescence) (Hashimoto et al., Brain Res.799:301-306, 1998; Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993).

Diseases and disorders that are associated with α-synuclein aggregatesare collectively referred to as synucleinopathies. Pathologically,α-synuclein has been identified as a major component of Lewy bodies, thehallmark inclusions of Parkinson's disease, and a fragment thereof wasisolated from amyloid plaques of a different neurological disease,Alzheimer's disease. Biochemically, recombinant α-synuclein has beenshown to form amyloid-like fibrils that recapitulated theultrastructural features of α-synuclein isolated from patients withdementia with Lewy bodies, Parkinson's disease and multiple systematrophy. Additionally, the identification of mutations within theα-synuclein gene, albeit in rare cases of familial Parkinson's disease,have demonstrated a strong link between synuclein pathology andneurodegenerative diseases. Thus, dysfunction of α-synuclein appears tobe a common link amongst the pathogenesis underlying a spectrum ofdiseases such as Parkinson's disease, dementia with Lewy bodies,multiple system atrophy and the Lewy body variant of Alzheimer'sdisease.

Fibrillization and aggregation of α-synuclein is thought to play majorrole in neuronal dysfunction and death of dopaminergic neurons inParkinson's disease. It has been suggested that mutations in α-synucleinor genomic triplication of wild type α-synuclein (leading to itsoverexpression) can cause certain rare familial forms of Parkinson'sdisease. A number of reports have indicated that over-expression ofwild-type α-synuclein induces neuronal cell death. See, e.g.,Polymeropoulos, et al. (1997) Science 276(5321):2045-7, Kruger, et al.(1998) Nat Genet. 18(2):106-8, Singleton, et al. (2003) Science302(5646):841, Miller, et al. (2004) Neurology 62(10):1835-8, Hashimoto,et al. (2003) Ann N Y Acad Sci. 991:171-88, Lo Bianco, et al. (2002)Proc Natl Acad Sci USA. 99(16):10813-8, Lee, et al. (2002) Proc NatlAcad Sci USA. 99(13):8968-73, Masliah, et al. (2000) Science 287(5456):1265-9, Auluck, et al. (2002) Science 295(5556):865-8, Oluwatosin-Chigbuet al. (2003) Biochem Biophys Res Commun 309(3): 679-84, Klucken et al.(2004) J Biol Chem. 279(24):25497-502. While it has been suggested thatprotecting neurons from the toxic effects of α-synuclein would be apromising strategy for treating Parkinson's disease and othersynucleinopathies such as Lewy body dementia, the exact targets relevantto the in vivo regulation of α-synuclein have been largely unknown.

At least three isoforms of synuclein are produced through alternativesplicing (Beyer K (September 2006).” Alpha-synuclein structure,posttranslational modification and alternative splicing as aggregationenhancers”. Acta Neuropathol. 112 (3): 237-51). The majority form of theprotein, and the one most investigated, is the full 140 amino acid-longpolypeptide, generally referred to as full-length α-synuclein. Otherisoforms are α-synuclein-126, where exon 3 is lost and lacks residues41-54; and α-synuclein-112 (Uéda K, Saitoh T, Mori H (December 1994).“Tissue-dependent alternative splicing of mRNA for NACP, the precursorof non-A beta component of Alzheimer's disease amyloid.”. Biochem.Biophys. Res. Commun. 205 (2): 1366-72), which lacks residue 103-130 dueto loss of exon 5 (Beyer K (September 2006). “Alpha-synuclein structure,posttranslational modification and alternative splicing as aggregationenhancers”. Acta Neuropathol. 112 (3): 237-51).

α-Synuclein is also known as SNCA. In humans, α-synuclein is encoded bythe SNCA gene. (Uéda K, Fukushima H, Masliah E, Xia Y, Iwai A, YoshimotoM, Otero D A, Kondo J, Ihara Y, Saitoh T (December 1993). “Molecularcloning of cDNA encoding an unrecognized component of amyloid inAlzheimer disease”. Proc. Natl. Acad. Sci. U.S.A. 90 (23): 11282-6; XiaY, Saitoh T, Uéda K, Tanaka S, Chen X, Hashimoto M, Hsu L, Conrad C,Sundsmo M, Yoshimoto M, Thal L, Katzman R, Masliah E (October 2001).“Characterization of the human alpha-synuclein gene: Genomic structure,transcription start site, promoter region and polymorphisms”. J.Alzheimers Dis. 3 (5): 485-494; Xia Y, Saitoh T, Uéda K, Tanaka S, ChenX, Hashimoto M, Hsu L, Conrad C, Sundsmo M, Yoshimoto M, Thal L, KatzmanR, Masliah E (2002). “Characterization of the human alpha-synucleingene: Genomic structure, transcription start site, promoter region andpolymorphisms: Erratum p 489 FIG. 3”. J. Alzheimers Dis. 4 (4): 337).

An α-synuclein fragment has been shown to be present in Alzheimer'sdisease amyloid. Originally identified as an unknown non-Abeta (or“non-Aβ”) component (NAC) in an amyloid-enriched fraction, this fragmentwas ultimately shown, through cloning of the full-length cDNA thatencodes it, to be a fragment of a precursor protein, known as NACP (UédaK, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero D A, KondoJ, Ihara Y, Saitoh T (December 1993). “Molecular cloning of cDNAencoding an unrecognized component of amyloid in Alzheimer disease”.Proc. Natl. Acad. Sci. U.S.A. 90 (23): 11282-6.doi:10.1073/pnas.90.23.11282. PMID 8248242. PMC 47966.http://www.pnas.org/content/90/23/11282). Subsequent to this cloning, itwas determined that NACP was the human homologue of Torpedo synuclein.Therefore, NACP is now referred to as human alpha-synuclein.

Alpha-synuclein is primarily found in neural tissue, making up to 1% ofall proteins in the cytosol (Iwai A, Masliah E, Yoshimoto M, Ge N,Flanagan L, de Silva H A, Kittel A, Saitoh T (February 1995). “Theprecursor protein of non-A beta component of Alzheimer's disease amyloidis a presynaptic protein of the central nervous system”. Neuron 14 (2):467-75). It is predominantly expressed in the neocortex, hippocampus,substantia nigra, thalamus, and cerebellum. It is predominantly aneuronal protein but can also be found in glial cells. In melanocyticcells, SNCA protein expression may be regulated by MITF (Hoek K S,Schlegel N C, Eichhoff O M, et al. (2008). “Novel MITF targetsidentified using a two-step DNA microarray strategy”. Pigment CellMelanoma Res. 21 (6): 665-76). It has been established thatalpha-synuclein is extensively localized in the nucleus of mammalianbrain neurons, suggesting a role of alpha-synuclein in the nucleus (YuS, Li X, Liu G, Han J, Zhang C, Li Y, Xu S, Liu C, Gao Y, Yang H, UédaK, Chan P (March 2007). “Extensive nuclear localization ofalpha-synuclein in normal rat brain neurons revealed by a novelmonoclonal antibody”. Neuroscience 145 (2): 539-55). Synuclein is,however, found predominantly in the presynaptic termini, in both free ormembrane-bound forms (McLean P J, Kawamata H, Ribich S, Hyman B T (March2000). “Membrane association and protein conformation of alpha-synucleinin intact neurons. Effect of Parkinson's disease-linked mutations”. J.Biol. Chem. 275 (12): 8812-6) with roughly 15% of synuclein beingmembrane-bound in any moment in neurons (Lee H J, Choi C, Lee S J(January 2002). “Membrane-bound alpha-synuclein has a high aggregationpropensity and the ability to seed the aggregation of the cytosolicform”. J. Biol. Chem. 277 (1): 671-8).

It has also been shown that alpha-synuclein localizes in neuronalmitochondria (Zhang L, Zhang C, Zhu Y, Cai Q, Chan P, Uéda K, Yu S, YangH (December 2008) “Semi-quantitative analysis of alpha-synuclein insubcellular pools of rat brain neurons: an immunogold electronmicroscopic study using a C-terminal specific monoclonal antibody”.Brain Res 1244: 40-52; Liu G, Zhang C, Yin J, Li X, Cheng F, Li Y, YangH, Uéda K, Chan P, Yu S (May 2009). “Alpha-Synuclein is differentiallyexpressed in mitochondria from different rat brain regions anddose-dependently down-regulates complex I activity”. Neurosci. Lett. 454(3): 187-92). Alpha-synuclein is highly expressed in the mitochondria inolfactory bulb, hippocampus, striatum, and thalamus, where the cytosolicalpha-synuclein is also rich; the cerebral cortex and cerebellum are twoexceptions, by contrast contain rich cytosolic alpha-synuclein but verylow levels of mitochondrial alpha-synuclein. Within the mitochondria, ithas been shown that alpha-synuclein is localized in the inner membraneof mitochondria, and that the inhibitory effect of alpha-synuclein oncomplex I activity of mitochondrial respiratory chain is dose-dependent.Thus, it is suggested that alpha-synuclein in mitochondria isdifferentially expressed in different brain regions and the backgroundlevels of mitochondrial alpha-synuclein may be a potential factoraffecting mitochondrial function and predisposing some neurons todegeneration.

It has been shown that alpha-synuclein significantly interacts withtubulin (Alim M A, Hossain M S, Arima K, Takeda K, Izumiyama Y, NakamuraM, Kaji H, Shinoda T, Hisanaga S, Uéda K. (January 2002). “Tubulin seedsalpha-synuclein fibril formation.”. J. Biol. Chem. 277 (3): 2112-7), andthat alpha-synuclein may have an activity as potentialmicrotubule-associated protein like tau (Alim M A, Ma Q L, Takeda K,Aizawa T, Matsubara M, Nakamura M, Asada A, Saito T, Kaji H, Yoshii M,Hisanaga S, Uéda K (August 2004). “Demonstration of a role foralpha-synuclein as a functional microtubule-associated protein”. J.Alzheimers Dis. 6 (4): 435-42; discussion 443-9).

Recent evidence suggests that alpha-synuclein functions as a molecularchaperone in the formation of SNARE complexes (Bonini N M, Giasson B I(November 2005). “Snaring the function of alpha-synuclein”. Cell 123(3): 359-61; Chandra S, Gallardo G, Fernández-Chacón R, Schlüter O M,Südhof T C (November 2005). “Alpha-synuclein cooperates with CSPalpha inpreventing neurodegeneration”. Cell 123 (3): 383-96). Indeed, there isgrowing evidence that alpha-synuclein is involved in the functioning ofthe neuronal Golgi apparatus and vesicle trafficking (A. A. Cooper, A.D. Gitler, A. Cashikar, C. M. Haynes, K. J. Hill, B. Bhullar, K. Liu, K.Xu, K. E. Strathearn, F. Liu, S. Cao, K. A. Caldwell, G. A. Caldwell, G.Marsischky, R. D. Kolodner, J. Labaer, J. C. Rochet, N. M. Bonini, andS. Lindquist. (2006). “Alpha-synuclein blocks ER-golgi traffic and Rab1rescues neuron loss in Parkinson's models”. Science 313 (5785):324-328).

Experimental evidence has been collected on the interaction ofalpha-synuclein with membrane and its involvement with membranecomposition and turnover. Yeast genome screening has found that severalgenes that deal with lipid metabolism play a role in alpha-synucleintoxicity (Willingham S, Outeiro T F, DeVit M J, Lindquist S L, MuchowskiP J (December 2003). “Yeast genes that enhance the toxicity of a mutanthuntingtin fragment or alpha-synuclein”. Science 302 (5651): 1769-72).Conversely, alpha-synuclein expression levels can affect the viscosityand the relative amount of fatty acids in the lipid bilayer (Uversky V N(October 2007). “Neuropathology, biochemistry, and biophysics ofalpha-synuclein aggregation”. J. Neurochem. 103 (1): 17-37).Alpha-synuclein is known to directly bind to lipid membranes,associating with the negatively charged surfaces of phospholipids(Uversky V N (October 2007). “Neuropathology, biochemistry, andbiophysics of alpha-synuclein aggregation”. J. Neurochem. 103 (1):17-37). A preferential binding to small vesicles has been found (Zhu M,Li J, Fink A L (October 2003). “The association of alpha-synuclein withmembranes affects bilayer structure, stability, and fibril formation”.J. Biol. Chem. 278 (41): 40186-97). The binding of alpha-synuclein tolipid membranes has complex effects on the latter, altering the bilayerstructure and leading to the formation of small vesicles (Madine J, DoigA J, Middleton D A (May 2006). “A study of the regional effects ofalpha-synuclein on the organization and stability of phospholipidbilayers”. Biochemistry 45 (18): 5783-92). Alpha-synuclein has beenshown to bend membranes of negatively charged phospholipid vesicles andform tubules from large lipid vesicles (Varkey J, Isas J M, Mizuno N,Jensen M B, Bhatia V K, Jao C C, Petrlova J, Voss J, Stamou D, Steven AC, Langen R (August 2010). “Membrane curvature induction and tubulationis a common feature of synucleins and apolipoproteins”. J Biol Chem).Studies have also suggested a possible antioxidant activity ofalpha-synuclein in the membrane (Zhu M, Qin Z J, Hu D, Munishkina L A,Fink A L (July 2006). “Alpha-synuclein can function as an antioxidantpreventing oxidation of unsaturated lipid in vesicles”. Biochemistry 45(26): 8135-42).

As described in more detail herein, the inventors of the instantdisclosure have discovered that α-synuclein is an in vivo target of theprotease caspase-1. Caspase-1 is a member of the cysteine proteasefamily of enzymes and is also commonly referred to as ICE. Caspase-1/ICEhas been widely studied for its involvement in the regulation ofapoptosis and inflammatory responses (e.g., cytokine production).Evidence presented herein shows that ICE cleaves the full-lengthα-synuclein polypeptide into at least two fragments, which are about 120amino acids and about 20 amino acids, respectively, and that theICE-mediated proteolytic site is localized toward the C-terminus of theα-synuclein polypeptide.

Based on the novel finding that α-synuclein is an in vivo substrate forthe ICE protease, the accompanying patent application entitled“ICE-cleaved α-synuclein as a biomarker” provides in more detail methodsdirected to use of ICE-cleaved fragments of α-synuclein as a marker fordetecting certain biological/clinical conditions.

α-Synuclein Antibodies

The invention also includes antibodies that are specific to α-synucleinfragments, including for example fragments which are the ICE-dependentcleavage products of the full-length α-synuclein. In some embodiments,the invention provides α-synuclein antibodies that specificallyrecognize α-synuclein that is not or has not been cleaved by ICE. It isknown that cysteine proteases including ICE catalyze the cleavage oftheir substrates following an aspartic acid residue (Asp or D) presenton the target. The α-synuclein primary sequence reveals that there arethree aspartic acid residues which are potential targets forICE-dependent proteolysis, which are at residues 115, 119 and 121 (shownin bold below).

Antibodies may be generated against a peptide based on the amino acidsequence of α-synuclein around residues 114-122 (114E, 115D, 116M, 117P,118V, 119D, 120P, 121D and 122N; shown with dotted underline above).Such antibodies can be generated by immunizing a laboratory animal withalpha-synuclein or a fragment thereof to induce antibodies, andscreening the resulting antibodies to identify those having the desiredbinding specificity. The antibody technology is highly developed and iswell known to one of ordinary skill in the art. Typically, an antigenicpeptide should contain at least 5-6 amino acid residues to conferspecificity. For example, in some embodiments, the antigenic peptideused to generate α-synuclein antibodies includes residues 114-116. Insome embodiments, the antigenic peptide includes residues 118-120. Insome embodiments, the antigenic peptide includes residues 120-122.

It is also possible to generate an antibody specific to the C-terminalfragment of α-synuclein corresponding generally to the last 20 aminoacid residues, such that the antibody will recognize and bind to bothfull-length α-synuclein and the ICE-cleaved α-synuclein of approximately20 amino acids.

Alternatively or additionally, α-synuclein antibodies may be generatedin accordance with the present invention against the N-terminus/centralportions of α-synuclein such that both the full-length and the larger(e.g., ˜120 amino acids) fragment of α-synuclein generated by ICEproteolysis can be detected.

α-Synuclein antibodies of the invention shall embrace, in addition tofull length immunoglobulins, various antigen-binding fragments thereof,which recognize full-length α-synuclein, and/or specific fragments ofα-synuclein generated by proteolysis.

The term “antibody” is used herein in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, antibody fragments, so long as they exhibitthe desired biological activity, and antibody like molecules such asscFv. A native antibody usually refers to heterotetrameric glycoproteinscomposed of two identical light (L) chains and two identical heavy (H)chains. Each heavy and light chain has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain at one end (VL) and a constant domain at its other end;the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

Certain portions of the variable domains differ extensively in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three or four segments called“complementarity-determining regions” (CDRs) or “hypervariable regions”in both in the light-chain and the heavy-chain variable domains. Themore highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four or five FR regions, largely adopting a β-sheetconfiguration, connected by the CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., NIH Publ. No,91-3242, Vol. I, pages 647-669 (1991)). The constant domains are notnecessarily involved directly in binding an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

A hypervariable region or CDR as used herein defines a subregion withinthe variable region of extreme sequence variability of the antibody,which form the antigen-binding site and are the main determinants ofantigen specificity. According to one definition, they can be residues(Kabat nomenclature) 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the lightchain variable region and residues (Kabat nomenclature 31-35 (H1), 50-65(H2), 95-102 (H3) in the heavy chain variable region. Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institute of Health, Bethesda, Md. [1991]). An“intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, CHI, CH2 and CH₃. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof. Preferably, the intact antibodyhas one or more effector functions. Various techniques have beendeveloped for the production of antibody fragments. Traditionally, thesefragments were derived via proteolytic digestion of intact antibodies(see, e.g., Morimoto et al., Journal of Biochemical and BiophysicalMethods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)).However, these fragments can now be produced directly by recombinanthost cells. For example, the antibody fragments can be isolated fromantibody phage libraries. Alternatively, Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (Carter et al., Bio/Technology 10:163-167 (1992)).

According to another approach, F(ab′)₂ fragments can be isolateddirectly from recombinant host cell culture. “Antibody fragments”comprise a portion of an intact antibody, preferably the antigen bindingor variable region of the intact antibody. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, each with asingle antigen-binding site, and a residual “Fc” fragment, whose namereflects its ability to crystallize readily. Pepsin treatment yields anF(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen. “Fv” is the minimum antibody fragmentwhich contains a complete antigen-recognition and -binding site. Thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. It is in this configurationthat the three CDRs of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDRs specific for an antigen) has the ability to recognize andbind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain which may be generated by papain digestion ofan intact antibody. The Fc region may be a native sequence Fc region ora variant Fc region. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue at aboutposition Cys226, or from about position Pro230, to the carboxyl-terminusof the Fc region. The FC region of an immunoglobulin generally comprisestwo constant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain. By “Fc region chain” herein is meant one of thetwo polypeptide chains of an Fc region.

The “hinge region,” and variations thereof, as used herein, includes themeaning known in the art, which is illustrated in, for example, Janewayet al., Immuno Biology: the immune system in health and disease(Elsevier Science Ltd., NY) (4th ed., 1999). Depending on the amino acidsequence of the constant domain of their heavy chains, immunoglobulinscan be assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond tothe different classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Methods of Identifying and/or Characterizing ICE Modulating Compounds

Further evidence presented herein implicates biological significance ofα-synuclein proteolysis by ICE. Thus, the invention embraces methods foridentifying and/or characterizing compounds that modulate α-synucleincleavage by ICE. In particular, the invention provides assays toidentify and/or characterize compounds that inhibit ICE.

It has been known for several years that Lewy Bodies, the aggregatesfound in the dying neurons of Parkinson's Disease (PD) patients,contain, in addition to ubiqutin and full-length α-synuclein, a fragmentof α-synuclein that appears to have been produced by specificproteolytic cleavage at around residue 120. Notably, several in vitrostudies have shown that this fragment aggregates more readily than thefull-length protein, leading a number of investigators to speculate thatthe fragment may nucleate aggregation in vivo (1). Inhibition of theproteolytic cleavage that produces the more toxic fragment wouldrepresent an attractive new strategy for preventing or arresting thedisease. However, until now, the identity of the in vivo enzyme(s)responsible for α-synuclein cleavage remained unknown. As described inExemplification below, the inventors of the instant application have forthe first time identified ICE to be at least one of the target enzymes,which can be inhibited to reduce the proteolytic cleavage ofα-synuclein.

Accordingly, featured herein are methods for screening for, identifying,and or characterizing a compound or compounds that inhibit ICE cleavageof α-synuclein. Such compounds can be used to treat a variety ofdiseases or conditions associated with abnormal α-synuclein processingand/or aggregation.

According to the invention, a provided method for identifying and/orcharacterizing an ICE inhibitor comprises the following steps: (1) aplurality of test compounds are provided, where the test compoundscontain candidate ICE inhibitor(s); (2) the test compounds are contactedwith full-length α-synuclein in the presence of ICE (e.g., in aproteolysis reaction); and (3) it is determined whether one or more ofthe test compounds inhibit ICE-dependent cleavage of the full-lengthα-synuclein.

Typically, relative degree of proteolysis of α-synuclein is determinedby measuring relative levels of full-length α-synuclein and cleavedα-synuclein in a reaction. A test compound which is an ICE inhibitor canreduce the amount of ICE-induced cleavage of α-synuclein under otherwiseidentical conditions. Therefore, a higher ratio of full-lengthα-synuclein to cleaved (fragment) α-synuclein in the presence of thetest compound as compared to a suitable control indicates that the testcompound is an ICE inhibitor that inhibits ICE cleavage of α-synuclein.“A suitable control” may be a compound known to be inert or otherwiseinactive as to modulating ICE activity, or may simply comprise thevehicle (e.g., reaction buffer) alone.

Relative levels of full-length and cleaved α-synuclein may be measuredby any suitable methods, such as protein immuno blotting (e.g., Westernblot) and mass spectrometry. A number of suitable techniques are knownto those skilled in the art.

A plurality of test compounds to be screened, identified, and/orcharacterized may comprise any variety of molecules. The term “compound”or “chemical compound” as used herein can include organometalliccompounds, organic compounds, metals, transitional metal complexes, andsmall molecules. In certain embodiments, polynucleotides are excludedfrom the definition of compounds. In certain embodiments,polynucleotides and peptides are excluded from the definition ofcompounds. In a particularly preferred embodiment, the term compoundsrefers to small molecules (e.g., preferably, non-peptidic andnon-oligomeric) and excludes peptides, polynucleotides, transition metalcomplexes, metals, and organometallic compounds.

Thus, candidate molecules may be one or more of a small molecule, apeptide, or a nucleic acid. The nucleic acids may be, for example, anRNA or DNA molecule, e.g., mRNA, RNAi, siRNA or an oligonucleotide.

“Small molecule”: As used herein, the term “small molecule” refers to anon-peptidic, non-oligomeric organic compound either synthesized in thelaboratory or found in nature. Small molecules, as used herein, canrefer to compounds that are “natural product-like”, however, the term“small molecule” is not limited to “natural product-like” compounds.Rather, a small molecule is typically characterized in that it containsseveral carbon-carbon bonds, and has a molecular weight of less than1500, although this characterization is not intended to be limiting forthe purposes of the present invention. Examples of “small molecules”that occur in nature include, but are not limited to, taxol, dynemicin,and rapamycin. Examples of “small molecules” that are synthesized in thelaboratory include, but are not limited to, compounds described in Tanet al., (“Stereoselective Synthesis of over Two Million Compounds HavingStructural Features Both Reminiscent of Natural Products and Compatiblewith Miniaturized Cell-Based Assays” J. Am. Chem. Soc. 120:8565, 1998;incorporated herein by reference). In certain other preferredembodiments, natural-product-like small molecules are utilized.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit proteindegradation in a cell, tissue or subject, or modulate a phenotype asdescribed herein of a cell, tissue or subject. The chemical librariescan be peptide libraries, peptidomimetic libraries, chemicallysynthesized libraries, recombinant, e.g., phage display libraries, andin vitro translation-based libraries, other non-peptide syntheticorganic libraries, etc.

Libraries screened using the methods of the present invention caninclude a variety of types of compounds. Examples of libraries that canbe screened in accordance with the methods of the invention include, butare not limited to, peptides; random biooligomers; diversomers such ashydantoins, benzodiazepines and dipeptides; vinylogous polypeptides;nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates;peptide nucleic acid libraries; antibody libraries; carbohydratelibraries; and small molecule libraries (preferably, small organicmolecule libraries). In some embodiments, the compounds in the librariesscreened are nucleic acid or peptide molecules. In a non-limitingexample, peptide molecules can exist in a phage display library. Inother embodiments, the types of compounds include, but are not limitedto, peptide analogs including peptides comprising non-naturallyoccurring amino acids, e.g., D-amino acids, phosphorous analogs of aminoacids, such as .gamma.-amino phosphoric acids and .gamma.-aminophosphoric acids, or amino acids having non-peptide linkages, nucleicacid analogs such as phosphorothioates and PNAs, hormones, antigens,synthetic or naturally occurring drugs, opiates, dopamine, serotonin,catecholamines, thrombin, acetylcholine, prostaglandins, organicmolecules, pheromones, adenosine, sucrose, glucose, lactose andgalactose. Libraries of polypeptides or proteins can also be used in theassays of the invention.

In certain embodiments, the combinatorial libraries are small organicmolecule libraries including, but not limited to, benzodiazepines,isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholinocompounds, and benzodiazepines. In another embodiment, the combinatoriallibraries comprise peptides; random bio-oligomers; benzodiazepines;diversomers such as hydantoins, benzodiazepines and dipeptides;vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates;peptidyl phosphonates; peptide nucleic acid libraries; antibodylibraries; or carbohydrate libraries. Combinatorial libraries arethemselves commercially available (see, e.g., ComGenex, Princeton, N.J.;Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow,Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia,Md.; etc.).

Compounds

Suitable compounds described herein for use according to the presentinvention include compounds incorporated herein by reference, andpharmaceutically acceptable derivatives thereof, that are particularlyeffective in the treatment and/or prevention of diseases, disorders,and/or conditions of the present invention. For instance, in someembodiments, described compounds are useful in the treatment and/orprevention of Parkinson's disease (including idiopathic Parkinson'sdisease (PD)), Diffuse Lewy Body Disease (DLBD) also known as Dementiawith Lewy Bodies (DLB), combined Alzheimer's and Parkinson diseaseand/or multiple system atrophy (MSA).

In some embodiments, described compounds for use in accordance with thepresent invention include any compound that inhibits ICE.

In some embodiments, described compounds are those that inhibit ICEselectively. In some embodiments, described compounds are those thatinhibit one or more enzymes in the caspase or ICE/CED-3 family inaddition or as an alternative to ICE.

In some embodiments, described compounds for use in accordance with thepresent invention include, but are not limited to, “WO 2005/117846”compounds. The phrase “WO 2005/117846 compounds” as used herein, refersto compounds as described and depicted in any one of the followingdocuments:

WO 03/068242, WO 03/042169, WO 98/16505, WO 93/09135, WO 03/106460, WO03/103677, WO 03/104231, WO 02/085899, WO 00/55114, WO 00/55127, WO00/61542, WO 01/05772, WO 01/10383, WO 01/16093, WO 01/42216, WO01/72707, WO 01/90070, WO 01/94351, WO 02/094263, WO 02/42278, U.S. Pat.No. 6,184,210, U.S. Pat. No. 6,184,244, U.S. Pat. No. 6,187,771, U.S.Pat. No. 6,197,750, U.S. Pat. No. 6,242,422, April 2001 AmericanChemical Society (ACS) Meeting in San Diego, Calif., USA, WO 02/22611,US 2002/0058630, WO 02/12638, WO 95/35308, U.S. Pat. No. 5,716,929, WO97/22619, U.S. Pat. No. 6,204,261, WO 99/47545, WO 01/90063, US PatentPublication 2004/0014753, US Patent Publication 2004/0009966, US PatentPublication 2003/0236296, US Patent Publication 2003/0096737, US PatentPublication 2003/0092703, US Patent Publication 2002/0169177, U.S. Pat.No. 6,693,096, U.S. Pat. No. 6,610,683, U.S. Pat. No. 6,531,467, U.S.Pat. No. 6,528,506, U.S. Pat. No. 6,200,969, WO 2003/072528, WO2003/032918, WO 01/00658, WO 98/10778, U.S. Pat. No. 6,716,818, U.S.Pat. No. 6,620,782, U.S. Pat. No. 6,566,338, U.S. Pat. No. 6,495,522,U.S. Pat. Nos. 6,355,618, 6,153,591, WO 2005/003100, WO 2004/002401, WO00/61542, WO 00/55114, WO 99/47154, U.S. Pat. No. 6,083,981, U.S. Pat.No. 5,932,549, U.S. Pat. No. 5,919,790, U.S. Pat. No. 5,744,451, WO2002/089749, WO 99/36426, WO 98/16505, WO 98/16504, WO 98/16502, U.S.Pat. No. 6,316,415, U.S. Pat. No. 5,932,549, U.S. Pat. No. 5,919,790,U.S. Pat. No. 5,744,451, EP 1082127, EP 1049703, EP 0932600, EP 0932598,WO 99/56765, WO 93/05071, EP 0600800 and EP 1378573 (which, as set forthherein, are all incorporated by reference herein). In one embodiment,compounds for use in this invention include those of WO 00/55114, WO00/55127, WO 00/61542, WO 00/61542, WO 01/05772, WO 01/10383, WO01/16093, WO 01/42216, WO 01/72707, WO 01/90070, WO 01/94351, USPublication 2003/0092703, WO 02/094263, US Publication 2002/0169177,U.S. Pat. No. 6,184,210, U.S. Pat. No. 6,184,244, U.S. Pat. No.6,187,771, U.S. Pat. No. 6,197,750, U.S. Pat. No. 6,242,422, April 2001American Chemical Society (ACS), meeting in San Diego, Calif., USA<WO02/22611, US Publication 2002/0058630, US Publication 2003/0096737, WO95/35308, WO 97/22619, WO 99/47545, and WO 01/90063. In anotherembodiment, compounds for use in this invention include those of WO04/058718, WO 04/002961, WO 95/35308, WO 97/22619, WO 99/47545, and WO01/90063. Alternately, compounds for use in this invention include thoseof WO 95/35308, WO 97/22619, WO 99/47545, and WO 01/90063. Preferredcompounds are those recited in the claims of the above-referenceddocuments. These compounds may be obtained by methods known to skilledpractitioners in the methods disclosed in documents cited herein.

In some embodiments, described compounds for use in accordance with thepresent invention include, but are not limited to, “Wannamaker”compounds. The phrase “Wannamaker compounds” as used herein, refers tocompounds as described and depicted in any one of the followingdocuments: U.S. Ser. No. 12/165,838, WO 91/15577, WO 93/05071, WO93/09135, WO 93/12076, WO 93/14777, WO 93/16710, WO 95/35308, WO96/30395, WO 9633209 and WO 98/01133; European patent applications 503,561, 547, 699, 618, 223, 623, 592, and 623 606, and U.S. Pat. Nos.5,434,248, 5,710,153, 5,716,929, 5,744,451, WO 95/26958; U.S. Pat. No.5,552,400; and Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996)(which, as set forth herein, are all incorporated by reference herein).

In some embodiments, described “Wannamaker” compounds for use inaccordance with the present invention are peptide and/or peptidylinhibitors of ICE.

In some embodiments, described “Wannamaker” compounds for use inaccordance with the present invention are non-peptidyl inhibitors ofICE.

In some embodiments, described “Wannamaker” compounds for use inaccordance with the present invention are inhibitors of ICE that arereported to have a favorable in vivo profile. Exemplary such compoundsinclude, but are not limited to, compounds of the following formula:

wherein the various substituents are as defined and described in U.S.Ser. No. 12/165,838 (now U.S. Pat. No. 8,022,041, the entirety of whichis herein incorporated by reference).

In some embodiments, described compounds for use in accordance with thepresent invention are pro-drugs of inhibitors of ICE including, but notlimited to, compounds as described and defined in U.S. Ser. No.12/165,838 (now U.S. Pat. No. 8,022,041, the entirety of which isincorporated herein by reference).

In some embodiments, described compounds for use in accordance with thepresent invention are of either of the following formulae:

In certain embodiments, described compounds for use in accordance withthe present invention are of either of the following formulae:

In certain embodiments, the compound is VX-765:

In some embodiments, the compound is NCGC00185682:

In some embodiments, described compounds for use in accordance with thepresent invention include, but are not limited to, “Zhang” compounds.The phrase “Zhang compounds” as used herein, refers to compounds asdescribed and depicted in Zhang et al., World J. Gastroenterol. 2007,13(4): 623-627 (the entirety of which is incorporated herein byreference). In some embodiments, a described “Zhang” compound for use inaccordance with the present invention isAc-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone.

In some embodiments, described compounds for use in accordance with thepresent invention include, but are not limited to, “Corasaniti”compounds. The phrase “Corasaniti compounds” as used herein, refers tocompounds as described and depicted in Corasaniti et al., Toxicol. Lett.2003, 4:139(2-3):213-9 (the entirety of which is incorporated herein byreference). In some embodiments, a described “Corasaniti” compound foruse in accordance with the present invention isAc-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) ort-butoxycarbonyl-L-aspartic acid benzyl ester chloromethylketone(Boc-Asp-(OBzl)-CMK).

In some embodiments, described compounds for use in accordance with thepresent invention include aspartic acid analogs as described and definedin WO 96/03982 (the entirety of which is incorporated herein byreference).

In some embodiments, described compounds are characterized in that theycause a detectable decrease (e.g., of at least an amount such as atleast 5%, at least 6%, at least 7%, at least 9%, at least 10%, at least11%, at least 12%, at least 13%, at least 14%, at least 15%, at least16%, at least 17%, at least 18%, at least 19%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or more) in the severityor frequency of one or more symptoms of the disease, disorder, orcondition of the present invention, and/or delay of onset of one or moresymptoms of a disease, disorder, or condition of the present invention.

In some embodiments, described compounds are characterized in that theycause a detectable change the levels of biomarkers associated with ICEinhibition.

In some embodiments, described compounds are characterized in that theycan inhibit or block pathophysiological effects of certain diseases asset forth herein.

In some embodiments, described compounds, by inhibiting ICE, directlyfacilitate the arrest or resolution of certain diseases describedherein, and/or facilitate the restoration of normal functioning.

In some embodiments, described compounds are characterized in that theyinhibit cleavage of a full-length α-synuclein polypeptide into two ormore fragments. In certain embodiments, described compounds arecharacterized in that the inhibit cleavage of a full length α-synucleinpolypeptide into at least two fragments having lengths of about 120amino acids and about 20 amino acids, or lengths of specifically 120amino acids and 20 amino acids.

In some embodiments, described compounds are characterized in that theyinhibit proteolytic α-synuclein cleavage. In certain embodiments,described compounds are characterized in that they inhibit proteolyticcleavage at or around a site corresponding to residue 120 in full lengthα-synuclein. In some embodiments, described compounds are characterizedin that they lessen the degree of proteolytic α-synuclein cleavage.

In some embodiments, described compounds are characterized in that theycause a higher ratio of full-length to cleaved fragments of α-synucleinin the cell as compared to control. In some embodiments, describedcompounds are characterized in that they cause a higher ratio offull-length to cleaved fragments of α-synuclein in the cell as comparedto control. In certain embodiments, a “higher ratio” is when the ratioof full-length to cleaved fragments of α-synuclein in a treated cell isone, two, three, four, five, six, seven, eight, nine, or ten timeshigher than as compared to the control. In certain embodiments, a“higher ratio” is when the ratio of full-length to cleaved fragments ofα-synuclein in a treated cell is at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% higher than as compared to the control.

In some embodiments, described compounds are characterized in that theyare capable of penetrating the blood-brain barrier (BBB) in atherapeutically effective amount. In some embodiments, describedcompounds are formulated as prodrugs, wherein the prodrug is capable ofpenetrating the blood-brain barrier (BBB), for example, in an amountgreater than that of the described compound when not in prodrug form. Insome embodiments, the prodrug passes readily through the blood brainbarrier. In certain embodiments, the prodrug has a brain penetrationindex of at least one, two, three, four, five, six seven, eight, nine,or ten times the brain penetration index of the drug alone. In someembodiments, the prodrug is stable in the environment of both thestomach and the bloodstream and may be delivered by ingestion.

In some embodiments, a prodrug comprises a hydrolyzable carrier. Once inthe central nervous system, the prodrug, which preferably is inactive,is hydrolyzed into the carrier and a provided compound or analog thereof(and optionally another drug). In some embodiments, the carrier is anormal component of the central nervous system and is inactive andharmless. The compound and/or drug, referred to herein as the “payload,”once released from the carrier, is active. In some embodiments, thecarrier is a fatty acid and comprises a partially-saturated straightchain molecule having between about 16 and 26 carbon atoms, and morepreferably 20 and 24 carbon atoms. Examples of fatty acid carriers areprovided in U.S. Pat. Nos. 4,939,174; 4,933,324; 5,994,932; 6,107,499;6,258,836; and 6,407,137, the disclosures of which are incorporatedherein by reference in their entirety.

In some embodiments, a described compound is a targeted compound. Asused herein, the phrase “targeted compound” refers to any compoundcomprising a targeting moiety and a payload. In some embodiments, atargeting moiety and payload are the same moiety and/or compound. Insome embodiments, a targeting moiety and payload are different moietiesand/or compounds. In some embodiments, a targeting moiety and payloadare encapsulated. In some embodiments, a targeting moiety and a payloadare covalently bound. In some embodiments, a targeting moiety and apayload are non-covalently bound. In some embodiments, a targetingmoiety and a payload are reversibly bound. In some embodiments, atargeting moiety and a payload are irreversibly bound. A “targeting”moiety, as used herein, is any moiety that facilitates delivery of apayload to a desired site with greater selectivity and/or specificityfor that site than would be achieved in the absence of the targetingmoiety. In some embodiments, a targeting moiety facilitates penetrationof the blood-brain barrier.

A “payload,” as used herein, is any one or more compounds used in thetreatment and/or prevention of diseases, disorders, and/or conditions ofthe present invention. In some embodiments, a payload is a WO2005/117846 compound. In some embodiments, a payload is a Wannamakercompound. In some embodiments, the Wannamaker compound is selected fromthe compounds disclosed in U.S. Ser. No. 12/165,838 (now U.S. Pat. No.8,022,041). In some embodiments, a payload is the Vertex prodrug VX-765,depicted below:

In some embodiments, a payload is a Zhang compound. In some embodiments,a payload is a Corasaniti compound. In some embodiments, a payload is anaspartic acid analog as described and defined in WO 96/03982. In someembodiments, a payload is the NIH compound NCGC00185682, depicted below:

In some embodiments, a payload is any one of the ICE inhibitorsdescribed and defined herein and/or incorporated by reference herein. Insome embodiments, a payload is an antioxidant. In some embodiments, apayload is an antioxidant that is capable of reducing oxidative stresssuch that activation of caspase-1 is inhibited.Compounds to be Screened, Identified, and/or Characterized

Compounds to be screened, identified, and/or characterized using one ormore methods described herein can be of any of a variety of chemicalclasses. In some embodiments, such compounds are small organic moleculeshaving a molecular weight in the range of 50 to 2,500 daltons. Suchcompounds can comprise functional groups involved in structuralinteraction with proteins (e.g., hydrogen bonding), and typicallyinclude at least an amine, carbonyl, hydroxyl, or carboxyl group, andpreferably at least two such functional chemical groups. Such compoundsoften comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures (e.g., purine core) substituted withone or more of the above functional groups.

In some embodiments, compounds are biomolecules such as, for example,polypeptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives or structural analogues thereof, polynucleotides, nucleicacid aptamers, polynucleotide analogs, carbohydrates, lipids, etc., orcombinations thereof. In some embodiments, compounds are antioxidants.In some embodiments, compounds are antioxidants and are screened for theability to reduce oxidative stress such that activation of caspase-1 isinhibited.

Compounds can be obtained or provided from any of a number of potentialsources, including: chemical libraries, natural product libraries, andcombinatorial libraries comprised of random peptides, oligonucleotides,or organic molecules. Chemical libraries consist of diverse chemicalstructures, some of which are analogs of known compounds or analogs orcompounds that have been identified as “hits” or “leads” in other drugdiscovery screens, while others are derived from natural products, andstill others arise from non-directed synthetic organic chemistry.Natural product libraries re collections of microorganisms, animals,plants, or marine organisms which are used to create mixtures forscreening by: (1) fermentation and extraction of broths from soil, plantor marine microorganisms, or (2) extraction of plants or marineorganisms. Natural product libraries include polypeptides, non-ribosomalpeptides, and variants (non-naturally occurring) thereof. For a review,see Science 282:63-68 (1998). Combinatorial libraries are composed orlarge numbers of peptides, oligonucleotides, or organic compounds as amixture. These libraries are relatively easy to prepare by traditionalautomated synthesis methods, PCR, cloning, or proprietary syntheticmethods. Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. In some embodiments, a chemical “library”contains only compounds that are structurally related to one another(e.g., share at least one common structural moiety; in many embodiments,a common core). In some embodiments, a chemical “library” contains aplurality, and in some embodiments, a majority of compounds that arestructurally related. In some embodiments, a chemical “library” containsa least one compound that is not structurally related (or notstructurally significantly related) to other compounds in the library.

For a review of combinatorial chemistry and libraries created therefrom,see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification oftest compounds through the use of the various libraries herein permitssubsequent modification of the test compound “hit” or “lead” to optimizethe capacity of the “hit” or “lead” to inhibit ICE in a mammalian cell.

Compounds for use in accordance with the present invention can besynthesized by any chemical or biological method. The compoundsidentified above can also be pure, or may be in a heterologouscomposition (e.g., a pharmaceutical composition), and can be prepared inan assay-, physiologic-, or pharmaceutically-acceptable diluent orcarrier as described in further detail herein (see PharmaceuticalCompositions and Methods of Treatment below).

Pharmaceutical compositions and methods provided herein are useful fortreating various conditions associated with ICE-dependent α-synucleinproteolysis.

Pharmaceutical compositions and methods provided herein are useful fortreating or preventing the various diseases, disorders, and conditionsas set forth herein.

The invention provides several screening methods to identify agentshaving a pharmacological activity useful in treating a synucleinopathy.The methods include screens that can be performed in vitro, in cells ortransgenic animals, and which test a variety of parameters as anindication of activity. Agents determined to have an activity in thesescreens can be retested in secondary screens of animal models ofsynucleinopathy or in clinical trials to determine activity againstbehavioral or other symptoms of these diseases.

As outlined below, the screening, identifying, and/or characterizingmethods contemplated herein include in vitro as well as in vivo (e.g.,cell and animal) assay systems. In some embodiments, a compound isconsidered to be an inhibitor if reduction of cleaved α-synuclein of atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more is observed inone or more assays as described herein. In some embodiments, a compoundis considered to be an inhibitor if reduction of cleaved α-synuclein ofat least 2, 3, 4, 5, 6, 7, 8, 9 or more times is observed in one or moreassays as described herein.

In Vitro Assays

Thus, in certain embodiments, enzymatic (e.g., proteolysis) assays arecarried out in vitro in which ICE cleavage of α-synuclein is measured inthe presence or absence of various test compounds. The assay isperformed in the presence of a specific concentration of the test agentor appropriate control under specific conditions. Specific conditionsinclude types of buffers, concentrations of agent, solvent agent isdissolved or suspended in, pH, temperature, time of incubation, etc.These particular parameters may be determined by the operator orscientist conducting the assay as would be appreciated by one of skillin this art. Modulatory effects of any test compound on α-synucleincleavage by ICE can be determined by measuring relative levels offull-length verses cleaved products of α-synuclein in the sample. Forexample, if in the presence of a test compound, there is less degree ofα-synuclein cleavage, then the test compound is a candidate inhibitor ofICE. In some embodiments, a candidate inhibitor of ICE is anantioxidant. In some embodiments, a candidate inhibitor of ICE is anantioxidant that inhibits activation of ICE.

Cell-Based Assays

In certain embodiments, ICE cleavage of α-synuclein may be assayed usingcellular systems. Thus, methods for identifying and/or characterizingcandidate compounds which may inhibit ICE cleavage of α-synuclein in acell comprise contacting a cell expressing α-synuclein and ICE with atest compound, and determining the modulatory effect of the testcompound on a phenotype of the cell with respect to the presence/levelsof full-length and cleaved products of α-synuclein, and/or aggregateformation of α-synuclein in the cell. In such methods, modulation of thephenotype is indicative of the efficacy of the compound. In particular,if the test compound causes a higher ratio of full-length to cleavedfragments of α-synuclein in the cell as compared to control, then thetest compound is a candidate inhibitor of ICE. In some embodiments, thephenotype of cells to be assayed includes measuring α-synucleinaggregate levels.

The cell types suitable for use in the cellular screening,identification, and/or characterization assays may be any cells thatexpress both α-synuclein and ICE. In some embodiments, either one orboth of α-synuclein and ICE are endogenously expressed. In otherembodiments, either one or both of α-synuclein and ICE are introducedinto the cells by transfection or infection of exogenous genes. In someembodiments, introduction of one or more exogenous genes or fragmentsthereof involves a transgenic animal model (discussed in further detailbelow). For example, cells suitable for described methods may beobtained from a transgenic animal source.

The contacting may be by adding the candidate compound to the media,directly to the cells, or as a fluid flowing over the cell, e.g., in alateral flow or a planar flow patch clamp device. One of skill in theart would be able to identify other appropriate methods having thebenefit of this disclosure.

Animal Models

In certain embodiments, the screening methods of the invention mayemploy one or more animal models. For example, transgenic miceexpressing various alleles of α-synuclein may be used to screen forcompounds that may inhibit ICE cleavage of α-synuclein in vivo. A numberof transgenic mouse lines that exhibit Parkinson's disease-likephenotype are commercially available. These include, without limitation,the following strains:

B6.129P2-Sncg^(tm1V1b)/J;

B6.Cg-Tg(THY1-SNCA*A53T)F53Sud/J;

B6.Cg-Tg(THY1-SNCA*A53T)M53Sud/J;

B6;129-Gt(ROSA)26Sor^(tm1(SNCA)*^(A53T)Djmo)/TmdJ;

B6;129-Gt(ROSA)26Sor^(tm2(SNCA)*^(119)Djmo)/TmdJ;

B6;129-Gt(ROSA)26Sor^(tm3(SNCA)*^(E46K)Djmo)/TmdJ;

B6;129X1-Snca^(tm1Ros1)/J;

B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J;

STOCK Tg(THY1-SNCA*A53T)F53Sud/J;

B6.129-Sncb^(tm1Sud)/J;

B6;129-Snca^(tm1Sud)Sncb^(tm1.1Sud)/J;

B6;SJL-Tg(THY1-SNCA*A30P)M30Sud/J;

C57BL/6-Tg(THY1-SNCA)1Sud/J; and,

STOCK Tg(THY1-Snca)M1mSud/J (available from Jackson Lab).

Methods for Identifying Other Enzymes

Processing of full-length α-synuclein to truncated fragments iscatalyzed by one or more proteases. Therefore, the present invention ina further aspect provides screening methods for identifying enzymes(e.g., proteases) that cleave α-synuclein in vitro and/or in vivo. Themethod used to identify ICE as an α-synuclein protease is described inthe Exemplification section below. The invention contemplatesidentifying additional proteases that cleave α-synuclein. Suchadditional proteases are candidates for therapeutic targets for thetreatment of Parkinson's disease and other α-synuclein-associateddiseases and conditions.

In certain embodiments, the method involves genetic screening toidentify a gene involved in α-synuclein processing. For example, thegene knock-down or knock-out technique can be employed to rescue acellular or systematic phenotype, such as cellular abnormalities orpathogenic features that can be detected. A number of model systems maybe suitable, including but are not limited to yeast and rodents, such asmice and rats.

In certain embodiments, the protease may be purified in vitro using asubstrate peptide (e.g., peptide inhibitor) identified by the screeningmethods discussed above. A preferred inhibitor is a peptide ofalpha-synuclein of e.g., at least about 5 but up to 20 contiguous aminoacids of full-length α-synuclein. In some embodiments, the peptideincludes residues 113, 114, 115 116, 117, 118, 119, 120, 121, 122, 123,124, 135, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139 and/or 140. In some embodiments, the peptide includes residues114-117, 111-126, 113-126, 113-119, 117-121 or 120-125, or 130-136,132-138, 131-135, 133-134, 133-137, or 135-136, in which a residueN-terminal to the cleavage site (e.g., between residues 115-116,119-120, 122-123, 133-134 and 135-136) has been replaced by a transitionstate analog. Such an inhibitor is used as an affinity purificationreagent to purify the protease from extracts of brain cells. Such cellscan be obtained from cadaver of a normal individual or one who hassuffered from a LBD disease. Levels of protease may be elevated in thelatter. The enzymatic activity of a protease can be assayed bypresenting it with an alpha-synuclein substrate and monitoring formationof cleavage products. End-specific antibodies described below are usefulfor detecting cleavage products. A substrate can be, for example, thenatural human form of alpha-synuclein described above, a fragmentthereof, containing residues flanking both sides of the cleavage site,or a mutant form thereof in which the mutation is associated with ahereditary form of LBD. Optionally, the C-terminus of the substrate canbe immobilized to a solid phase, and the N-terminus to a label. Cleavageof a substrate releases the label to a liquid phase. The liquid phasecan readily be separated from the solid phase, and the amount of labelquantified as a measure of proteolytic activity.

Therapy

Based on the finding that ICE cleaves α-synuclein to generate fragmentsthat are more prone to aggregate to form Lewy Bodies (LB), the inventionprovides methods for treating a subject suffering from or at risk ofdeveloping at least one form of synucleinopathies. Provided methodscomprise administering to the subject an effective amount of ICEinhibitor to inhibit α-synuclein cleavage so as to reduce the formationof toxic α-synuclein fragments in cells.

The term “synucleinopathy,” as used herein, refers to a disease,disorder or condition associated with abnormal expression, stability,activities and/or cellular processing of α-synuclein. Thus the termembraces so-called Lewy Body Disease (LBD) which is characterized bydegeneration of the dopaminergic system, motor alterations, cognitiveimpairment, and formation of Lewy bodies (LBs). (McKeith et al.,Clinical and pathological diagnosis of dementia with Lewy bodies (DLB):Report of the CDLB International Workshop, Neurology (1996) 47:1113-24).Lewy Bodies are spherical protein deposits found in affected nervecells. Their presence in the brain disrupts the brain's normal functioninterrupting the action of neurotransmitters including acetylcholine anddopamine. Synucleinopathies include Parkinson's disease (includingidiopathic Parkinson's disease (PD)), Diffuse Lewy Body Disease (DLBD)also known as Dementia with Lewy Bodies (DLB), Combined Alzheimer's andParkinson disease and multiple system atrophy (MSA). DLBD sharessymptoms of both Alzheimer's and Parkinson's disease (includingParkinson's disease chemically induced by exposure to environmentalagents such as pesticides, insecticides, or herbicides and/or metalssuch as manganese, aluminum, cadmium, copper, or zinc, SNCA gene-linkedParkinson's disease, sporadic or idiopathic Parkinson's disease, orParkin- or LRRK2-linked Parkinson's disease). DLBD differs fromParkinson's disease mainly in the location of Lewy Bodies. In DLBD LewyBodies form mainly in the cortex. In Parkinson's disease, they formmainly in the substantia nigra. Other synucleinopathies include PureAutonomic Failure, Lewy body dysphagia, Incidental LBD, Inherited LBD(e.g., mutations of the alpha-synuclein gene, PARK3 and PARK4), andMultiple System Atrophy (e.g., Olivopontocerebellar Atrophy,Striatonigral Degeneration and Shy-Drager Syndrome).

Thus, the compositions and methods described herein are useful fortreating various conditions associated with ICE-dependent α-synucleinproteolysis.

Target Populations

Subjects who are candidates for an ICE inhibitor therapy according tothe present invention may be at present symptomatic or asymptomatic ofone or more forms of synucleinopathies. In some embodiments, a candidatesubject (e.g., patient) has been diagnosed with at least one form ofsynucleinopathies, such as Parkinson's disease.

In some embodiments, a candidate subject (e.g., patient) has not beendiagnosed with synucleinopathies but is considered at risk of developingat least one form of synucleinopathies. For example, a subject may carrya genetic allele that renders him or her susceptible to such a disease.In some cases, a subject's family history may indicate the risk.

In some embodiments, a patient is free of clinical symptoms or riskfactors of any amyloidogenic disease other than one characterized byLewy bodies. In some embodiments, a patient is free of clinical symptomsor risk factors of any disease characterized by extracellular amyloiddeposits. In some embodiments, a patient is free of diseasescharacterized by amyloid deposits of A-beta peptide. In someembodiments, a patient is free of clinical symptoms and risk factors ofAlzheimer's disease. In some methods, a patient has concurrentAlzheimer's disease and a disease characterized by Lewy bodies. In someembodiments, a patient has concurrent Alzheimer's and Parkinson'sdisease.

In some embodiments, a candidate subject for receiving an ICE inhibitortherapy described herein for the treatment of synucleinopathy is notbeing treated for a known inflammatory condition, where the ICEinhibitor is administered for purposes of inhibiting pro-inflammatorycytokine production or signaling. Common inflammatory conditions forwhich an ICE inhibitor is administered for purposes of inhibitingpro-inflammatory cytokines include arthritis, asthma and other allergicconditions. The most common cellular ICE targets (substrates) for theseconditions include cytokines, such as precursors of IL-1β and IL-18,which promote the Th2 immunity upon cleavage by ICE and therefore arepro-inflammatory.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30, etc.). Usually, however, it is not necessary to begin treatmentuntil a patient reaches 40, 50, 60 or 70. Treatment typically entailsmultiple dosages over a period of time. Effectiveness of a treatment canbe evaluated by determining a subject's responsiveness to the treatment.In some embodiments, it may be monitored by assaying relative amounts offull-length and cleaved α-synuclein proteins in a biological samplecollected from the subject (e.g., patient). In certain embodiments,detecting the mere presence of certain α-synuclein fragments (cleavageproducts) in a biological sample may be indicative of a pathologicalcondition. In some embodiments, levels of antibody, or activated T-cellor B-cell responses to a therapeutic agent (e.g., a truncated form ofalpha-synuclein peptide) may be monitored over time. In someembodiments, two or more parameters are combined to confirm diagnosis orresponsiveness to a therapy over time.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of asynucleinopathy in a regime comprising an amount and frequency ofadministration of the composition or medicament sufficient to eliminateor reduce the risk, lessen the severity, or delay the outset of thedisease, including physiological, biochemical, histologic and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease. Intherapeutic applications, compositions or medicates are administered toa patient suspected of, or already suffering from such a disease in aregime comprising an amount and frequency of administration of thecomposition sufficient to cure, or at least partially arrest, thesymptoms of the disease (physiological, biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of the disease. An amount adequate toaccomplish therapeutic or prophylactic treatment is defined as atherapeutically- or prophylactically-effective dose. A combination ofamount and dosage frequency adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically orprophylactically-effective regime. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

In some embodiments, administration of an agent results in reduction ofintracellular levels of aggregated alpha-synuclein. In some methods,administration of the agent results in a reduction in levels ofC-terminal truncated forms of alpha-synculein. In some methods,administration of an agent results in improvement in a clinical symptomof a synucleinopathy, such as motor or cognitive function in the case ofParkinson's disease. In some methods, reduction in intracellular levelsof aggregated alpha-synuclein or improvement in a clinical symptom ofdisease is monitored at intervals after administration of an agent.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

As provided further below, compounds described herein can optionally beadministered in combination with other agents that are at least partlyeffective in treatment of synucleinopathy. Compounds of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.

Administration

The term “administration” or “administering” includes routes ofintroducing the compound of the invention(s) to a subject to performtheir intended function. Examples of routes of administration that maybe used include injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations may be given by formssuitable for each administration route. For example, these preparationsare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, suppository, etc., administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred. The injection can bebolus or can be continuous infusion. Depending on the route ofadministration, the compound of the invention can be coated with ordisposed in a selected material to protect it from natural conditionswhich may detrimentally effect its ability to perform its intendedfunction. The compound of the invention can be administered alone, or inconjunction with either another agent as described above or with apharmaceutically-acceptable carrier, or both. The compounds of theinvention can be administered prior to the administration of the otheragent, simultaneously with the agent, or after the administration of theagent. Furthermore, the compound of the invention can also beadministered in a pro-form which is converted into its activemetabolite, or more active metabolite in vivo.

The language “biological activities” of a compound of the inventionincludes all activities elicited by compound of the inventions in aresponsive cell. It includes genomic and non-genomic activities elicitedby these compounds.

The term “effective amount” as used herein includes an amount effective,at dosages and for periods of time necessary, to achieve the desiredresult, e.g., sufficient to treat a disorder. An effective amount ofcompound of the invention may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound of the invention to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of the compound of theinvention are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of compound of the invention (e.g.,an effective dosage) may range from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a compound of the invention can include a singletreatment or, preferably, can include a series of treatments. In oneexample, a subject is treated with a compound of the invention in therange of between about 0.1 to 20 mg/kg body weight, one time per weekfor between about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of a compound of the invention used for treatment may increase ordecrease over the course of a particular treatment.

Combination Therapy

It is further contemplated that the treatment method comprising an ICEinhibitor described herein may be used in combination with one or moreadditional therapeutics for the treatment of synucleinopathy, such thatthe ICE inhibitor is administered to a subject in conjunction with asynucleinopathy therapy other than an ICE inhibitor. Additionaltherapeutic agents that are normally administered to treat a particulardisease or condition may be referred to as “agents appropriate for thedisease, or condition, being treated.”

“In conjunction with” means that the ICE inhibitor and additionaltherapy or therapies are administered to a subject in combination. Theadministrations may be simultaneous administration or separateadministrations.

Thus, in some embodiments of the present invention, compounds describedherein may be administered in combination with one or more additionaltherapeutic agents. Such additional therapeutic agents may beadministered separately from a described compound-containingcomposition, as part of a multiple dosage regimen. Alternatively oradditionally, such agents may be part of a single dosage form, mixedtogether with a described compound in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the terms “combination,” “combined,” and related termsrefer to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a describedcompound may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a described compound, anadditional therapeutic agent, and a pharmaceutically acceptable carrier,adjuvant, or vehicle. Two or more agents are typically considered to beadministered “in combination” when a patient or individual issimultaneously exposed to both agents. In many embodiments, two or moreagents are considered to be administered “in combination” when a patientor individual simultaneously shows therapeutically relevant levels ofthe agents in a particular target tissue or sample (e.g., in brain, inserum, etc.).

The amount of both a described compound and additional therapeutic agent(in those compositions which comprise an additional therapeutic agent asdescribed above) that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. Preferably, compositions inaccordance with the invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of a described compound can beadministered.

In some embodiments of the invention, agents that are utilized incombination may act synergistically. Therefore, the amount of eitheragent utilized in such situations may be less than that typicallyutilized or required in a monotherapy involving only that therapeuticagent. Commonly, a dosage of between 0.01-1,000 μg/kg body weight/day ofthe additional therapeutic agent can be administered.

The amount of additional therapeutic agent present utilized incombination therapy according to the present invention typically will beno more than the amount that would normally be administered in acomposition comprising that therapeutic agent as the only active agent.Preferably the amount of additional therapeutic agent utilized willrange from about 50% to 100% of the amount normally utilized intherapies involving that agent as the only therapeutically active agent.Established dosing regimens for known therapeutic agents are known inthe art and incorporated herein by reference.

For example, compounds described herein, or pharmaceutically acceptablecompositions thereof, can be administered in combination with one ormore treatments for Parkinson's Disease such as L-DOPA/carbidopa,entacapone, ropinrole, pramipexole, bromocriptine, pergolide,trihexephendyl, and amantadine; For example, methods of the presentinvention can be used in combination with medications for treating PD.Such therapeutic agents include levodopa, carbodopa, levodopa (Sinemetand Sinemet CR), Stalevo (carbodopa, levodopa, and entacapone),anticholinergics (trihexyphenidyl, benztropine mesylate, procyclidine,artane, cogentin), bromocriptidine (Parlodel), pergolide (Permax),ropinirol (Requip), pramipexole (Mirapex), cabergoline (Dostinex),apomorphine (Apokyn), rotigotine (Neupro), Ergolide, Mirapex or Requip.

In some embodiments, described compositions and formulations may beadministered in combination with one or more treatments for Parkinson'sDisease such as ACR-343, rotigotine (Schwarz), rotigotine patch (UCB),apomorphine (Amarin), apomorphine (Archimedes), AZD-3241 (Astra Zeneca),creatine (Avicena), AV-201 (Avigen), lisuride (Axxonis/Biovail),nebicapone (BIAL Group), apomorphine (Mylan), CERE-120 (Ceregene),melevodopa+carbidopa (Cita Neuropharmaceuticals), piclozotan (Daiichi),GM1 Ganglioside (Fidia Farmaceutici), Altropane (Harvard University),Fluoratec (Harvard University), fipamezole (Juvantia Pharma),istradefylline (Kyowa Hakko Kogyo), GPI-1485 (MGI GP), Neu-120 (NeurimPharmaceuticals), NGN-9076 (NeuroGeneration Inc), NLX-P101 (Neurologix),AFQ-056 (Novartis), arundic acid (Ono/Merck & Co), COMT inhibitor(Orion), ProSavin (Oxford Biomedica), safinamide (Pharmacia & Upjohn),PYM-50028 (Phytopharm), PTX-200 (Phytix), 123I-iometopane (ResearchTriangle Institute), SYN-115 (Roche Holding), preladenant (ScheringPlough), ST-1535 (Sigma-Tau Ind. Farm), ropinirole (SmithKline Beecham),pardoprunox (Solvay), SPN-803 (Supernus Pharmaceuticals), nitisinone(Syngenta), TAK-065 (Takeda), cell therapy (Titan Pharmaceuticals), PDgene therapy (University of Auckland/Weill Medical College), 18F-AV-133(University of Michigan), mitoquinone/mitoquinol redox mixture(Antipodean Pharmaceuticals), 99m-Tc-tropantiol (University ofPennsylvania), apomorphine (Vectura), BIIB-014 (Vernalis Group),aplindore (Wyeth), and XP-21279 (XenoPort Inc).

Alternatively or additionally, in some embodiments, describedcompositions and formulations may be administered in combination withone or more treatments for Alzheimer's disease such as Aricept® andExcelon®. In some embodiments, described compositions and formulationsmay be administered in combination with one or more treatments forParkinson's Disease such as ABT-126(Abbott Laboratories), pozanicline(Abbott Laboratories), MABT-5102A (AC Immune), Affitope AD-01 (AFFiRiSGmbH), Affitope AD-02 (AFFiRiS GmbH), davunetide (Allon TherapeuticsInc), nilvadipine derivative (Archer Pharmaceuticals), Anapsos (ASACPharmaceutical International AIE), ASP-2535 (Astellas Pharma Inc),ASP-2905 (Astellas Pharma Inc), 11C-AZD-2184 (AstraZeneca plc),11C-AZD-2995 (AstraZeneca plc), 18F-AZD-4694 (AstraZeneca plc), AV-965(Avera Pharmaceuticals Inc), AVN-101 (Avineuro Pharmaceuticals Inc),immune globulin intravenous (Baxter International Inc), EVP-6124 (BayerAG), nimodipine (Bayer AG), BMS-708163 (Bristol-Myers Squibb Co),CERE-110 (Ceregene Inc), CLL-502 (CLL Pharma), CAD-106 (CytosBiotechnology AG), mimopezil ((Debiopharm SA), DCB-AD1 (DevelopmentCentre for Biotechnology), EGb-761 ((Dr Willmar Schwabe GmbH & Co),E-2012 (Eisai Co Ltd), ACC-001(Elan Corp plc), bapineuzumab (Elan Corpplc), ELND-006(Elan Pharmaceuticals Inc), atomoxetine (Eli Lilly & Co),LY-2811376 (Eli Lilly & Co), LY-451395 (Eli Lilly & Co), m266 (Eli Lilly& Co), semagacestat (Eli Lilly & Co), solanezumab (Eli Lilly & Co),AZD-103 (Ellipsis Neurotherapeutics Inc), FGLL (ENKAM PharmaceuticalsA/S), EHT-0202 (ExonHit Therapeutics SA), celecoxib (GD Searle & Co),GSK-933776A (GlaxoSmithKline plc), rosiglitazone XR (GlaxoSmithKlineplc), SB-742457 (GlaxoSmithKline plc), R-1578 (Hoffmann-La Roche AG),HF-0220 (Hunter-Fleming Ltd), oxiracetam (ISF Societa Per Azioni),KD-501 (Kwang Dong Pharmaceutical Co Ltd), NGX-267 (Life ScienceResearch Israel), huperzine A (Mayo Foundation), Dimebon (MedivationInc), MEM-1414 (Memory Pharmaceuticals Corp), MEM-3454 (MemoryPharmaceuticals Corp), MEM-63908 (Memory Pharmaceuticals Corp), MK-0249(Merck & Co Inc), MK-0752 (Merck & Co Inc), simvastatin (Merck & CoInc), V-950 (Merck & Co Inc), memantine (Merz & Co GmbH), neramexane(Merz & Co GmbH), Epadel (Mochida Pharmaceutical Co Ltd), 123I-MNI-330(Molecular Neuroimaging Lk), gantenerumab (MorphoSys AG), NIC5-15 (MountSinai School of Medicine), huperzine A (Neuro-Hitech Inc), OXIGON (NewYork University), NP-12 (Noscira SA), NP-61 (Noscira SA), rivastigmine(Novartis AG), ECT-AD (NsGene A/S), arundic acid (Ono Pharmaceutical CoLtd), PF-3084014 (Pfizer Inc), PF-3654746 (Pfizer Inc), RQ-00000009(Pfizer Inc), PYM-50028 (Phytopharm plc), Gero-46 (PN Gerolymatos SA),PBT-2 (Prana Biotechnology Ltd), PRX-03140 (Predix Pharmaceuticals Inc),Exebryl-1 (ProteoTech Inc), PF-4360365 (Rinat Neuroscience Corp), HuCALanti-beta amyloid monoclonal antibodies (Roche AG), EVT-302 (RocheHolding AG), nilvadipine (Roskamp Institute), galantamine (SanochemiaPharmazeutika AG), SAR-110894 (sanofi-aventis), INM-176 (Scigenic &Scigen Harvest), mimopezil (Shanghai Institute of Materia Medica of theChinese Academy of Sciences), NEBO-178 (Stegram Pharmaceuticals),SUVN-502 (Suven Life Sciences), TAK-065 (Takeda Pharmaceutical),ispronicline (Targacept Inc), rasagiline (Teva PharmaceuticalIndustries), T-817MA (Toyama Chemical), PF-4494700 (TransTech PharmaInc), CX-717 (University of California), 18F-FDDNP (University ofCalifornia Los Angeles), GTS-21 (University of Florida), 18F-AV-133(University of Michigan), 18F-AV-45 (University of Michigan),tetrathiomolybdate (University of Michigan), 123I-IMPY (University ofPennsylvania), 18F-AV-1/ZK (University of Pennsylvania), 11C-6-Me-BTA-1(University of Pittsburgh), 18F-6-OH-BTA-1 (University of Pittsburgh),MCD-386 (University of Toledo), leuprolide acetate implant (VoyagerPharmaceutical Corp), aleplasinin (Wyeth), begacestat (Wyeth), GSI-136(Wyeth), NSA-789 (Wyeth), SAM-531 (Wyeth), CTS-21166 (Zapaq), andZSET-1446 (Zenyaku Kogyo).

Alternatively or additionally, in some embodiments, describedcompositions and formulations may be administered in combination withone or more treatments for motor neuronal disorders, such as AEOL-10150(Aeolus Pharmaceuticals Inc), riluzole (Aventis Pharma AG), ALS-08(Avicena Group Inc), creatine (Avicena Group Inc), arimoclomol (BiorexResearch and Development Co), mecobalamin (Eisai Co Ltd), talampanel(Eli Lilly & Co), R-7010 (F Hoffmann-La Roche Ltd), edaravone(Mitsubishi-Tokyo Pharmaceuticals Inc), arundic acid (Ono PharmaceuticalCo Ltd), PYM-50018 (Phytopharm plc), RPI-MN (ReceptoPharm Inc), SB-509(Sangamo BioSciences Inc), olesoxime (Trophos SA), sodium phenylbutyrate(Ucyclyd Pharma Inc), and R-pramipexole (University of Virginia).

Alternatively or additionally, in some embodiments, describedcompositions and formulations may be administered in combination withone or more antioxidants. In some embodiments, described compositionsand formulations may be administered in combination with one or moreantioxidants capable of reducing oxidative stress such that activationof caspase-1 is inhibited. Exemplary antioxidants are known in thechemical and medicinal arts and are identified using methods describedabove and herein.

Pharmaceutical Compositions

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

In some embodiments, the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of a described compound, formulated together with one ormore pharmaceutically acceptable carriers (additives) and/or diluentsfor use in treating Parkinson's disease (including idiopathicParkinson's disease (PD)), Diffuse Lewy Body Disease (DLBD) also knownas Dementia with Lewy Bodies (DLB), Combined Alzheimer's and Parkinsondisease, multiple system atrophy (MSA), or any other diseases,disorders, or conditions associated with α-synuclein. As described indetail, pharmaceutical compositions of the present invention may bespecially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

Pharmaceutically acceptable salts of compounds described herein includeconventional nontoxic salts or quaternary ammonium salts of a compound,e.g., from non-toxic organic or inorganic acids. For example, suchconventional nontoxic salts include those derived from inorganic acidssuch as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric,nitric, and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like.

In other cases, described compounds may contain one or more acidicfunctional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. These salts can likewise be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. See, for example,Berge et al., supra.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations for use in accordance with the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, and the particular mode of administration. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, this amount will range fromabout 1% to about 99% of active ingredient, preferably from about 5% toabout 70%, most preferably from about 10% to about 30%.

In certain embodiments, a formulation as described herein comprises anexcipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a described compound of the presentinvention.

Methods of preparing formulations or compositions comprising describedcompounds include a step of bringing into association a compound of thepresent invention with the carrier and, optionally, one or moreaccessory ingredients. In general, formulations may be prepared byuniformly and intimately bringing into association a compound of thepresent invention with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations described herein suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. Compounds described hereinmay also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), an active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glycerol; disintegrating agents, such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; solution retarding agents, such asparaffin; absorption accelerators, such as quaternary ammoniumcompounds; wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; absorbents, such as kaolin andbentonite clay; lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

Tablets may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

Tablets and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may alternatively or additionallybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be formulatedfor rapid release, e.g., freeze-dried. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Dosage forms for topical or transdermal administration of a compound ofthis invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Such compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Inclusion ofone or more antibacterial and/or and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like, may bedesirable in certain embodiments. It may alternatively or additionallybe desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it may bedesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution, which in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe described compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In certain embodiments, a described compound or pharmaceuticalpreparation is administered orally. In other embodiments, a describedcompound or pharmaceutical preparation is administered intravenously.Alternative routs of administration include sublingual, intramuscular,and transdermal administrations.

When compounds described herein are administered as pharmaceuticals, tohumans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1% to 99.5% (more preferably,0.5% to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Preparations described herein may be given orally, parenterally,topically, or rectally. They are of course given in forms suitable forthe relevant administration route. For example, they are administered intablets or capsule form, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion or inhalation;topical by lotion or ointment; and rectal by suppositories. Oraladministrations are preferred.

Such compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, compounds describedherein which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the invention may be varied so as to obtain an amount ofthe active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of described compounds employed in the pharmaceuticalcomposition at levels lower than that required to achieve the desiredtherapeutic effect and then gradually increasing the dosage until thedesired effect is achieved.

In some embodiments, one or more described compounds, or pharmaceuticalcompositions thereof, is provided to a synucleinopathic subjectchronically. Chronic treatments include any form of repeatedadministration for an extended period of time, such as repeatedadministrations for one or more months, between a month and a year, oneor more years, or longer. In many embodiments, chronic treatmentinvolves administering one or more described compounds, orpharmaceutical compositions thereof, repeatedly over the life of thesubject. Preferred chronic treatments involve regular administrations,for example one or more times a day, one or more times a week, or one ormore times a month. In general, a suitable dose such as a daily dose ofone or more described compounds, or pharmaceutical compositions thereof,will be that amount of the one or more described compound that is thelowest dose effective to produce a therapeutic effect. Such an effectivedose will generally depend upon the factors described above. Generallydoses of the compounds of this invention for a patient, when used forthe indicated effects, will range from about 0.0001 to about 100 mg perkg of body weight per day. Preferably, the daily dosage will range from0.001 to 50 mg of compound per kg of body weight, and even morepreferably from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

If desired, the effective daily dose of one or more described compoundsmay be administered as two, three, four, five, six, or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a described compound to be administered alone,it is preferable to administer a described compound as a pharmaceuticalformulation (composition) as described above.

Described compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine, by analogy withother pharmaceuticals.

According to the invention, described compounds for treatingneurological conditions or diseases can be formulated or administeredusing methods that help the compounds cross the blood-brain barrier(BBB). The vertebrate brain (and CNS) has a unique capillary systemunlike that in any other organ in the body. The unique capillary systemhas morphologic characteristics which make up the blood-brain barrier(BBB). The blood-brain barrier acts as a system-wide cellular membranethat separates the brain interstitial space from the blood.

The unique morphologic characteristics of the brain capillaries thatmake up the BBB are: (a) epithelial-like high resistance tight junctionswhich literally cement all endothelia of brain capillaries together, and(b) scanty pinocytosis or transendothelial channels, which are abundantin endothelia of peripheral organs. Due to the unique characteristics ofthe blood-brain barrier, hydrophilic drugs and peptides that readilygain access to other tissues in the body are barred from entry into thebrain or their rates of entry and/or accumulation in the brain are verylow.

In one aspect of the invention, described compounds that cross the BBBare particularly useful for treating synucleinopathies. In oneembodiment, described compounds that cross the BBB are particularlyuseful for treating Parkinson's Disease (PD). Therefore it will beappreciated by a person of ordinary skill in the art that some of thecompounds of the invention might readily cross the BBB. Alternatively,the compounds of the invention can be modified, for example, by theaddition of various substituents that would make them less hydrophilicand allow them to more readily cross the BBB.

Various strategies have been developed for introducing those drugs intothe brain which otherwise would not cross the blood-brain barrier.Widely used strategies involve invasive procedures where the drug isdelivered directly into the brain. One such procedure is theimplantation of a catheter into the ventricular system to bypass theblood-brain barrier and deliver the drug directly to the brain. Theseprocedures have been used in the treatment of brain diseases which havea predilection for the meninges, e.g., leukemic involvement of the brain(U.S. Pat. No. 4,902,505, incorporated herein in its entirety byreference).

Although invasive procedures for the direct delivery of drugs to thebrain ventricles have experienced some success, they are limited in thatthey may only distribute the drug to superficial areas of the braintissues, and not to the structures deep within the brain. Further, theinvasive procedures are potentially harmful to the patient.

Other approaches to circumventing the blood-brain barrier utilizepharmacologic-based procedures involving drug latentiation or theconversion of hydrophilic drugs into lipid-soluble drugs. The majorityof the latentiation approaches involve blocking the hydroxyl, carboxyland primary amine groups on the drug to make it more lipid-soluble andtherefore more easily able to cross the blood-brain barrier.

Another approach to increasing the permeability of the BBB to drugsinvolves the intra-arterial infusion of hypertonic substances whichtransiently open the blood-brain barrier to allow passage of hydrophilicdrugs. However, hypertonic substances are potentially toxic and maydamage the blood-brain barrier.

Antibodies are another method for delivery of compositions of theinvention. For example, an antibody that is reactive with a transferrinreceptor present on a brain capillary endothelial cell, can beconjugated to a neuropharmaceutical agent to produce anantibody-neuropharmaceutical agent conjugate (U.S. Pat. No. 5,004,697,incorporated herein in its entirety by reference). Such methods areconducted under conditions whereby the antibody binds to the transferrinreceptor on the brain capillary endothelial cell and theneuropharmaceutical agent is transferred across the blood brain barrierin a pharmaceutically active form. The uptake or transport of antibodiesinto the brain can also be greatly increased by cationizing theantibodies to form cationized antibodies having an isoelectric point ofbetween about 8.0 to 11.0 (U.S. Pat. No. 5,527,527, incorporated hereinin its entirety by reference).

A ligand-neuropharmaceutical agent fusion protein is another methoduseful for delivery of compositions to a host (U.S. Pat. No. 5,977,307,incorporated herein in its entirety by reference). The ligand isreactive with a brain capillary endothelial cell receptor. The method isconducted under conditions whereby the ligand binds to the receptor on abrain capillary endothelial cell and the neuropharmaceutical agent istransferred across the blood brain barrier in a pharmaceutically activeform. In some embodiments, a ligand-neuropharmaceutical agent fusionprotein, which has both ligand binding and neuropharmaceuticalcharacteristics, can be produced as a contiguous protein by usinggenetic engineering techniques. Gene constructs can be preparedcomprising DNA encoding the ligand fused to DNA encoding the protein,polypeptide or peptide to be delivered across the blood brain barrier.The ligand coding sequence and the agent coding sequence are inserted inthe expression vectors in a suitable manner for proper expression of thedesired fusion protein. The gene fusion is expressed as a contiguousprotein molecule containing both a ligand portion and aneuropharmaceutical agent portion.

The permeability of the blood brain barrier can be increased byadministering a blood brain barrier agonist, for example bradykinin(U.S. Pat. No. 5,112,596, incorporated herein in its entirety byreference), or polypeptides called receptor mediated permeabilizers(RMP) (U.S. Pat. No. 5,268,164, incorporated herein in its entirety byreference). Exogenous molecules can be administered to the host'sbloodstream parenterally by subcutaneous, intravenous or intramuscularinjection or by absorption through a bodily tissue, such as thedigestive tract, the respiratory system or the skin. The form in whichthe molecule is administered (e.g., capsule, tablet, solution, emulsion)depends, at least in part, on the route by which it is administered. Theadministration of the exogenous molecule to the host's bloodstream andthe intravenous injection of the agonist of blood-brain barrierpermeability can occur simultaneously or sequentially in time. Forexample, a therapeutic drug can be administered orally in tablet formwhile the intravenous administration of an agonist of blood-brainbarrier permeability is given later (e.g., between 30 minutes later andseveral hours later). This allows time for the drug to be absorbed inthe gastrointestinal tract and taken up by the bloodstream before theagonist is given to increase the permeability of the blood-brain barrierto the drug. On the other hand, an agonist of blood-brain barrierpermeability (e.g., bradykinin) can be administered before or at thesame time as an intravenous injection of a drug. Thus, the term“co-administration” is used herein to mean that the agonist ofblood-brain barrier and the exogenous molecule will be administered attimes that will achieve significant concentrations in the blood forproducing the simultaneous effects of increasing the permeability of theblood-brain barrier and allowing the maximum passage of the exogenousmolecule from the blood to the cells of the central nervous system.

In other embodiments, a described compound can be formulated as aprodrug with a fatty acid carrier (and optionally with anotherneuroactive drug). The prodrug is stable in the environment of both thestomach and the bloodstream and may be delivered by ingestion. Theprodrug passes readily through the blood brain barrier. The prodrugpreferably has a brain penetration index of at least two times the brainpenetration index of the drug alone. Once in the central nervous system,the prodrug, which preferably is inactive, is hydrolyzed into the fattyacid carrier and a described compound or analog thereof (and optionallyanother drug). The carrier preferably is a normal component of thecentral nervous system and is inactive and harmless. The compound and/ordrug, once released from the fatty acid carrier, is active. Preferably,the fatty acid carrier is a partially-saturated straight chain moleculehaving between about 16 and 26 carbon atoms, and more preferably 20 and24 carbon atoms. Examples of fatty acid carriers are provided in U.S.Pat. Nos. 4,939,174; 4,933,324; 5,994,932; 6,107,499; 6,258,836; and6,407,137, the disclosures of which are incorporated herein by referencein their entirety.

Administration of agents of the present invention may be for eitherprophylactic or therapeutic purposes. When provided prophylactically,the agent is provided in advance of disease symptoms. The prophylacticadministration of the agent serves to prevent or reduce the rate ofonset of symptoms of Parkinson's disease (including idiopathicParkinson's disease (PD)), Diffuse Lewy Body Disease (DLBD) also knownas Dementia with Lewy Bodies (DLB), Combined Alzheimer's and Parkinsondisease and multiple system atrophy (MSA). When providedtherapeutically, the agent is provided at (or shortly after) the onsetof the appearance of symptoms of actual disease. In some embodiments,the therapeutic administration of the agent serves to reduce theseverity and duration of the disease.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (e.g., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl. Compositions for parenteral administration aretypically substantially sterile, substantially isotonic and manufacturedunder GMP conditions of the FDA or similar body.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein. Alternatively, transdermal delivery canbe achieved using a skin path or using transferosomes (Paul et al., Eur.J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta1368, 201-15 (1998)).

EXEMPLIFICATIONS

Provided below is an exemplary embodiment of the present invention,demonstrating inhibition of caspase-1/ICE as a promising therapeuticapproach to treat Parkinson's disease. It should not be construed to belimiting in any way.

Background

The protein α-synuclein is associated with multiple neurologicaldisorders, including the two most prevalent neurodegenerative diseases,Parkinson disease and Alzheimer disease. Collectively, these α-synucleinassociated disorders are referred to as synucleinopathies, and most arecharacterized by the presence of insoluble α-synuclein-rich aggregatescalled Lewy bodies (1-3). The presence of Lewy bodies in neurons of thesubstantia nigra is the histopathological hallmark of Parkinson disease,and is currently used to differentiate Parkinson disease from otherneurological disorders with overlapping clinical symptoms (4). Inaddition to α-synuclein being the major component of Lewy bodies foundin the sporadic form of Parkinson disease (4), monogenic point mutations(A30P, A53T, and E46K) as well as gene duplication and triplication ofthe α-synuclein locus have been identified as causal factors of earlyonset familial Parkinson disease (5-7). As such, α-synuclein is likelyinvolved in a pathogenic pathway common to both sporadic and familialforms of synucleinopathies. The role of α-synuclein in normal brainfunction is still poorly understood. There is evidence that it plays arole in synaptic vesicle transport and possibly in mitochondrial fusionand fission; it is also important for memory and learning in mice andsong birds, respectively (8, 9). Overexpression of human α-synuclein inyeast and C. elegans (neither of which expresses α-synuclein naturally)results in defective ER-Golgi vesicular transport, a result ofderegulation of the Rab1 GTPase (3, 10). α-synuclein is small (140residues) and highly conserved in vertebrates. Its sequence containsmultiple KTKE (SEQ ID NO: 3) or EKTK (SEQ ID NO: 4) imperfect amino acidrepeats spanning the first 2/3 of the protein (residues 1 to 83), whilethe C-terminal region (residues 100-140) is highly acidic. The repeatsegments have high α-helical propensity and helical structure isdetected by circular dichroism (CD) and nuclear magnetic resonance (NMR)when α-synuclein is incubated with some detergents and lipid vesicles(11, 12).

It has been known for several years that Lewy Bodies, the aggregatesfound in the dying neurons of Parkinson's Disease (PD) patients,contain, in addition to ubiquitin and full-length α-synuclein, afragment of α-synuclein that appears to have been produced by specificproteolytic cleavage at around residue 120. Several in vitro studieshave shown that this fragment aggregates more readily than thefull-length protein, leading a number of investigators to speculate thatthe fragment may nucleate aggregation in vivo (1). Inhibition of theproteolytic cleavage that produces the fragment would represent anattractive new strategy for preventing or arresting the disease.However, there are hundreds of proteases in the human genome and manyare essential genes; as such, it has not been possible to identify thetarget enzyme.

To identify the target enzyme(s) responsible for cleaving α-synuclein invivo, we turned to yeast. In contrast to the situation in mammaliancells, yeast has fewer than 60 proteases and none is an essential gene.Yeast also has no brain, which one would think might make it a poormodel organism for PD research, but Lindquist's lab showed thatoverexpression of human α-synuclein in yeast resulted in aggregation andcytotoxicity, and went on to show that genes that suppressed thistoxicity when overexpressed along with synuclein could suppresssynuclein toxicity in mouse and cell culture models of Parkinson'sDisease (2). These and other observations suggested to us that yeastmight represent a model organism capable of simplifying the proteasehunt, so we set out first to find the protease in yeast and then tovalidate that enzyme in human cells as a PD target.

Example 1 Toxic α-Synuclein Fragments

The present invention demonstrated that the aggregates formed in yeastwhen human α-synuclein is overexpressed contain the same fragment of theprotein that is found in Lewy Bodies.

Example 2 Demonstration of the Involvement of Certain Cysteine Proteases

Next, each of the more than 50 yeast proteases in thesynuclein-overexpression strain were systematically deleted in order tosee if any of the deletions rescued yeast from synuclein toxicity. Twodeletions did: deletion of RIM13, the sole yeast homologue of the humancysteine protease calpain, and YCA1, the sole yeast homologue of thehuman caspase family of cysteine proteases. Since in yeast RIM13activates YCA1, it seemed possible that only a single enzyme wascleaving synuclein in yeast. Loss of either protease not only abolishedsynuclein toxicity, it also eliminated the production of synucleinfragments and aggregates. The involvement of cysteine proteases wasconfirmed by screening a battery of protease inhibitors to see if any ofthem would prevent synuclein toxicity in yeast. Only the non-specificcysteine protease inhibitors were effective.

Humans have 28 calpain and caspase isozymes—a large but not impossiblenumber to test. RNAi was used to knock down each of these in turn in aneuronal cell culture model of PD. It is a neuroblastoma cell line(BE(2)-M17) carrying a wild-type α-synuclein overexpressing vector.BE(2)-M17 is a clone of the SK-N-BE(2) cell line that was established inNovember of 1972 from a bone marrow biopsy taken from a 2 year old boywith neuroblastoma. Synuclein on its own is not toxic to these cells,but it is toxic when combined with an oxidative stress agent such asrotenone or menadione. Greater than 90% reduction of mRNA levels foreach of the 29 protease genes was routinely achieved, and greater than50% reduction in protein levels (typically, about 70% reduction) wasroutinely achieved.

Example 3 Identification of ICE as a Target Protease

The present example demonstrates that ICE is a target protease. It wasdetermined that in this cell culture model not only did synuclein formLewy Body-like aggregates, but it also gave rise to the same fragmentfound in yeast and PD brains. Each of the knockdown cell lines wereexamined for the loss of this fragment, and it was found that reductionin the amount of only one of the human calpains and caspases abolishedfragment production: caspase-1.

Caspase-1 is not an essential gene. Moreover, there is a crystalstructure already known for this protein, which is sometimes referred toas interleukin-1-beta converting enzyme (ICE). It is expressed in brain,including the neurons of the nigra pars compacta. Due to its importancein inflammation, caspase-1 was investigated thoroughly by several drugcompanies as a possible target for the treatment of chronic inflammatorydiseases. A number of very potent, highly specific inhibitors of theenzyme were developed (3). Although none of those drugs reached themarket, at least two, from Vertex Corporation, passed Phase 1 clinicaltrials and were determined to be safe for use in humans. None of thesedrugs have ever been tested as a possible treatment for Parkinson'sDisease.

Example 4 Confirmation that ICE Cleaves Alpha-Synuclein In Vitro

The present invention further demonstrates that purified caspase-1/ICEcan be used to verify that caspase-1/ICE cleaves α-synuclein in vitro,and produces the expected fragmentation. Purified, activated caspase-1does indeed cleave alpha-synuclein (FIG. 5). In this set of in vitroassays, a 60 ul reaction mixture consisting of 21 ug of synuclein,various amount of ICE in 100 mM HEPES, pH 7.4, 0.1% OG, 10% glycerol,150 mM NaCl. Incubated at 37° C. for 2 hours. 20 ul were withdrawn fromthe reaction for SDS-PAGE followed by western blotting withanti-synuclein antibody (FIG. 5).

Moreover, the caspase-1 inhibitor from the NIH lab completely blocksthis cleavage in a dose-dependent manner (FIG. 6), demonstrating itsspecificity. Here, a 60 ul reaction mixture consisting of 21 ug ofsynuclein, 10 ug ICE in 100 mM HEPES, pH 7.4, 0.1% OG, 10% glycerol, 150mM NaCl, with or without 20 uM inhibitor was incubated at 37° C. for 2hours. 20 ul were withdrawn from the reaction for SDS-PAGE followed bywestern blotting with anti-synuclein antibody (FIG. 6). As shown in FIG.4, activity against a model substrate (Ac-YVAD-AMC, from Enzo, Inc.)(SEQ ID NO: 5) was determined to be 110 uM/min/mg. Moreover,fragmentation of alpha-synuclein in vitro was shown to be ICE specific(FIG. 6).

Mass spectrometer was used to determine the site of cleavage. There wasonly one cut, at residue 121, exactly as is observed in the fragmentfound in Lewy Bodies. The fragment generated by ICE was determined to be13167 Da which corresponds to residues from 1-121 (FIG. 7). For massspectrometry experiments, a 60 ul reaction mixture consisting of 21 ugof synuclein, 10 ug ICE in 100 mM HEPES, pH 7.4, 0.1% OG, 10% glycerol,150 mM NaCl. Incubated at 37° C. for 2 hours. Mixed with matrix andanalyzed with MALDI-TOF mass spec. A representative datum is shown inFIG. 7.

Example 5 Confirmation that ICE Cleaves Alpha-Synuclein In Vivo

The present example confirms that ICE cleaves alpha-synuclein in vivo.While the in vivo assays have been more challenging due to persistentcaspase-1 activation at low levels in unstressed cells (possibly becausethe cells are not really unstressed), it is definitive that caspase-1cleaves alpha-synuclein both in vitro and in vivo and thatcaspase-1-cleaved alpha-synuclein aggregates much faster thanfull-length, wild type alpha-synuclein (e.g., SEQ ID NO: 1).

Example 6 Demonstration that Oxidative Stress Activates ICE to CleaveAlpha-Synuclein

The present example demonstrates, for the first time, that oxidativestress at the mitochondria can activate caspase-1 and induce cleavage ofalpha-synuclein, thereby providing a possible explanation for the knownPD-inducing agents such as rotenone and MPTP (FIG. 3). In this set ofexperiments, M17 overexpressing α-synuclein was grown in Optimem inpresence or absence of menadione to confluence. Cells were then scrapedand lysed by sonication for SDS-PAGE followed by western blotting withanti-synuclein antibody.

Menadione was shown to promote alpha-synuclein induced toxicity in aneuronal cell line (M17). It was found that low concentrations ofmenadione are not toxic to M17 cells carrying empty pcDNA3 vector, butare toxic to M17 cells overexpressing a-synuclein (FIG. 8 and FIG. 9).

To further support the biological significance of ICE on α-synucleinprocessing, known inhibitors of ICE may be utilized. For example, Vertexprovides ICE inhibitors, which are tested for their inhibitory activitythat can block fragment formation in vitro.

It may be further tested that the ICE inhibitor, e.g., the Vertexcompounds, can block synuclein fragment formation in the Cooksonneuroblastoma model of PD, or any other suitable PD model systems, suchas transgenic animal models. Additionally, it may be examined that thissame compound rescues yeast from α-synuclein toxicity.

Example 7 Demonstration of Ability of RNAi and Chemical Inhibition ofICE to Reduce Alpha-Synuclein Fragmentation in Nerve Cells

A cell viability assay was performed using LIVE/DEAD (molecular probes)assay according to the manufacture instructions. As summarized in FIG.10, both RNAi knockdown of caspase-1/ICE and treatment with thecaspase-1/ICE inhibitor block synuclein fragment formation in nervecells and should inhibit synuclein aggregation in neuronal cells derivedfrom one or more mouse models of PD.

It is contemplated that once it has been determined that inhibition ofcaspase-1 is protective in neurons derived from PD mice, it is thentested that same inhibitor on the mice themselves. The ability of an ICEinhibitor (e.g., the Vertex compound) to penetrate the blood-brainbarrier in animals may be also examined using well-known techniques suchas mass spectrometry, a technique routinely used to measure drugpenetration into the brain.

It would be necessary to determine the suitable dosing protocol, andthere would need to be several end-points, including survival andreduction or abolition of fragment production. A crucial question isjust what effect one should be looking for. If the fragment nucleatesthe production of toxic synuclein oligomers, but does not effect thekinetics of aggregate propagation, it is likely that inhibition ofcaspase-1/ICE would delay the onset of disease but may not completelyprevent it. In certain situations, inhibiting the protease may affectdisease progression once PD symptoms have already appeared. Thus,caspase-1/ICE inhibition is a potential treatment for diagnosed PD, aswell as a possible way to prevent or delay disease initiation. In sum,the above examples confirm that (1) caspase-1 cleaves alpha-synuclein invitro and in vivo at the site found in the fragments of alpha-synuclein(α-Syn) that are observed in Lewy Bodies; and (2) RNAi knockdown orchemical inhibition of caspase-1 reduces fragment formation in nervecells in culture. These results demonstrate that caspase-1 (ICE)inhibition appears to be a valid and promising target for Parkinson's(PD) therapy.

Example 8 Compounds Screening

Yet further contemplated is to screen additional (novel) ICE inhibitorcompounds with preferred pharmacokinetics with respect to brainpenetration and half-life. A number of diversity libraries of smallmolecules with known or likely ability to cross the blood-brain barrierare available and may be used for these screening

In some cases, in silico screening using the already determined crystalstructure may be employed.

In addition, screening may be done by direct genetic/functionalscreening in our yeast model for synuclein toxicity.

Example 9 Pretreatment with Caspase-1 Inhibitors

The present Example describes an approach for assessing the ability ofpretreatment with caspase-1 inhibitors to prevent or delay theaccumulation and aggregation of aSyn expression and improve neurologicalfunctional recovery in rotenone-treated rats. In particular, the presentExample determines whether caspase-1 inhibition will prevent or delayaSyn fragment formation and aggregation and prevent motor disability inthe rotenone mouse PD model. Of particular interest is the Vertexprodrug VX-765 and the NIH compound NCGC00185682.

Research Design

Subjects: A total of 50 C57/BL mice comprise this experiment.

Experimental procedure: Seven month old male Lewis rats (300-350 g) areemployed. For the pretreatments experiments (Groups 1-2), rats receivecaspase-1 inhibitors (50 mg/kg based on similarities to the Vertexcompound) or vehicle for one week. Then, Groups 1-2 (above) receive 3mg/kg rotenone once per day for 10 days. This is a dosing paradigm thatproduces motor impairments, loss of nigrostriatal dopamine, and alphasynuclein aggregation (13) (Groups 5 and 6 receive the caspase-1inhibitor or vehicle followed by vehicle in lieu of the rotenone. Thisallows for assessment of the effects of caspase-1 inhibitors upon thenormal animal and determination of whether caspase-1 treated animalsexhibit structural and functional neuroprotection to the level of normalanimals (vehicle-vehicle). Daily caspase-1 inhibitor or vehicletreatment continues for 14 days following the rotenone (or vehicle)treatment. A this point, the model becomes stable. From this time pointforward, motor function using the rotorod, rearing (apomorphinestimulated and not) and postural instability tests are evaluated.Animals are sacrificed two months after the last rotenone injection forhistological and biochemical analysis.

Outcome measures are: 1) behavioral data from the rotarod test, rearingand postural instability tests; 2) stereological counts of nigral DAT-and TH-ir neurons and aSyn-ir cells in both aggregated andnon-aggregated forms within the SN; 3) HPLC measurements of dopamine andits metabolites, 4) measurement of the optical density of TH-ir withinstriatum; 5) Western Blot Analysis for aSyn protein expression andfragment formation; and 6) Quantitative RT-PCR analysis for aSyn mRNAexpression within SN.

Group Number Group Description Total Numbers of Animals 1 caspase-1inhibitor 10 (histological (left side of the brain) pretreatment + andbiochemical (right side rotenone of the brain) analysis) 2 vehiclepretreatment + 10 (histological (left side of the brain) rotenone andbiochemical (right side of the brain) analysis) 3 caspase-1 inhibitor 10(histological (left side of the brain) posttreatment + and biochemical(right side rotenone of the brain) analysis) 4 vehicle posttreatment +10 (histological (left side of the brain) rotenone and biochemical(right side of the brain) analysis) 5 caspase-1 inhibitor + 5(histological (left side of the brain) vehicle and biochemical (rightside of the brain) analysis) 6 vehicle + vehicle 5 (histological (leftside of the brain) and biochemical (right side of the brain) analysis)

Example 10 Treatment with Caspase-1 Inhibitors

The present Example describes an approach for determining the ability ofcaspase-1 inhibitor treatment delivered after rotenone treatment toreverse or retard the fragmentation and aggregation of alpha-synucleinand thus improve neurological functional recovery in therotenone-treated rat.

Experiment 1b: All aspects of this experiment are identical toExperiment 1a above, except that the caspase-1 inhibitor is administeredonly at days 7-14 post-rotenone (or vehicle) to determine whether thistreatment can reverse an already established synucleinopathy.

Example 11 Prevention with Caspase-1 Inhibitors

The present Example describes an approach for determining the ability ofcaspase-1 inhibitor treatment to prevent or delay the fragmentation andaggregation of aSyn and improve neurological functional recovery in ratsreceiving intranigral viral over-expression of alpha synuclein.

Experiment 2: Experiment 2 tests the hypothesis that a caspase-1inhibitor (at ˜50 mg/kg) reduces the aSyn fragmentation and aggregationwithin the SN and improves functional recovery in the AAV6-aSyn treatedrat PD model. The AAV vector empty plasmid is commercially available(pAAV-MCS) from Stratagene. Full human wild type aSyn gene (includingcoding region+3′UTR) are cloned into the AAV6 plasmid.

Research Design:

Subjects: 100 young adult Sprague Dawley male rats comprise thisexperiment.

Experimental procedure: For one week, rats receive daily injections ofcaspase-1 inhibitors or vehicle. Then rats in groups 1-4 above receivevector injections comprised of 2 ul of equally titered AAV6-alpha syn orAAV6-GFP. Caspase-1 treatment is also tested in rats receivingintranigral injection of vehicle due to the potential general toxicityof these vectors (especially GFP) for which caspase-1 inhibitortreatment may have effects. All rats continue to receive caspase-1 orvehicle treatment for 6 weeks, after which time they are sacrificed forhistological and biochemical analysis. Statistical analysis is similarto Experiment 1 above.

Outcome measures are: Same as Experiment 1 except analysis of behavioraldata of motor function will be performed on the cylinder test.

Statistical Analysis: Behavioral, neuroanatomical, neurochemical, andmolecular measures comparisons between different groups are assessedusing a factorial ANOVA. Post-hoc tests controlling for multiplecomparisons are employed to test individual group differences.

Group Number Group Description Total Numbers of Animals 1 caspase-1inhibitor + 20 (10 for histological analysis and 10 AAV6 alpha-synucleinfor biochemical analysis) 2 vehicle + AAV6 alpha- 20 (10 forhistological analysis and 10 synuclein for biochemical analysis) 3caspase-1 inhibitor + 20 (10 for histological analysis and 10 AAV6alpha-synuclein for biochemical analysis) 4 vehicle + AAV6-GFP 20 (10for histological analysis and 10 for biochemical analysis) 5 caspase-1inhibitor + 10 (5 for histological analysis and 5 vehicle forbiochemical analysis) 6 vehicle + vehicle 10 (5 for histologicalanalysis and 5 for biochemical analysis)

Outcomes: It is contemplated that caspase-1 inhibition will 1) reducethe fragmentation and aggregation of aSyn protein within the SN; 2)delay or block nigrostriatal dopaminergic degeneration, and 3) preventor delay the onset of motor disability in one or both rat models of PD.

One aspect of this experiment design is that it relies on theadministration of a toxin to generate the PD-like model. A supplementalprotocol is therefore contemplated to include testing in analpha-synuclein transgenic model of PD. Also considered is the use of analpha-synuclein knockout mouse as a negative control to address the roleof alpha-synuclein in these studies. In some embodiments, experimentaldesigns that incorporate alpha-synuclein transgenic (and knockout mice)include protocol wherein doses of Caspase I inhibitor are varied.

Example 12 Blood-Brain Barrier (BBB) Penetration

The present example describes an approach for assessing is the abilityof, inter alia, the two compounds referenced above (i.e., VX-765 andNCGC00185682) to cross the blood brain-barrier (BBB). Preliminary testssuggest that BBB penetration in animals is low for both compounds. Inview of the fact that studies of VX-765 as a potential epilepsytherapeutic are currently underway at Vertex, it is hypothesized thateither BBB penetration in humans is better or that the amount ofcompound in the CNS that is needed to achieve the desired effect beingmeasured in the studies is small.

The following two-stage process may be used to determine BBBpermeability.

(1) Cell-Free Permeability

Collagen-coated, microporous, polycarbonate membranes in 12-well CostarTranswell® plates without MDR1-MDCK cells were used for this study. Thepermeability assay buffer was Hanks Balanced Salt Solution containing 10mM HEPES with 15 mM glucose at a pH of 7.4. The dosing solutionconcentration of the test compounds was 1 μM in the assay buffer. Induplicate, cell-free inserts were dosed on the top (apical) chamber andincubated at 37° C. with 5% CO₂ in a humidified incubator. After 120minutes, aliquots were taken from the receiver chambers. Samples weretaken from the donor chamber at 5 and 120 minutes. All samples wereassayed by LC-MS/MS. The apparent permeability, P_(app), and percentrecovery were calculated as follows:

P _(app)=(dCr/dt)×Vr/(A×C ₀)  (1)

Percent Recovery=100×((Vr×C _(rfinal))+(Vd×C _(dfinal)))/(Vd×C_(N))  (2)

Where, dCr/dt is the slope of the cumulative concentration in thereceiver compartment versus time in μM/s; Vr is the volume of thereceiver compartment in cm³; Vd is the volume of the donor compartmentin cm³; A is the area of the insert (1.13 cm² for 12-well Transwell®);C₀ is the measured 0 minute donor concentration in 1 μM; C_(N) is thenominal concentration of the dosing solution in 1 μM; C_(rfinal) is thecumulative receiver concentration in 1 μM at the end of the incubationperiod; C_(dfinal) is the concentration of the donor in 1 μM at the endof the incubation period.

(2) Permeability, MDR1-MDCK

MDR1-MDCK monolayers were grown to confluence on collagen-coated,microporous, polycarbonate membranes in 12-well Costar Transwell®plates. Details of the plates and their certification are shown below.The permeability assay buffer was Hanks Balanced Salt Solutioncontaining 10 mM HEPES and 15 mM glucose at a pH of 7.4 The dosingsolution concentration was 5 μM in the assay buffer. Cell monolayerswere dosed on the apical side (A-to-B) or basolateral side (B-to-A) andincubated at 37° C. with 5% CO₂ in a humidified incubator. At 60 and 120minutes, aliquots were taken from the receiver chambers and replacedwith fresh assay buffer. Samples were taken from the donor chamber at120 minutes. Each determination was performed in duplicate. The luciferyellow flux was measured for each monolayer after being subjected to thetest compounds to ensure no damage was inflicted to the cell monolayersduring the flux period. All samples were assayed by LC-MS/MS usingelectrospray ionization. The apparent permeability, P_(app), and percentrecovery were calculated as follows:

P _(app)=(dCr/dt)×Vr/(A×C _(N))  (1)

Percent Recovery=100×((Vr×C _(rfinal))+(Vd×C _(dfinal)))/(Vd×C_(N))  (2)

Where dCr/dt is the slope of the cumulative concentration in thereceiver compartment versus time in μM/s; Vr is the volume of thereceiver compartment in cm³; Vd is the volume of the donor compartmentin cm³; A is the area of the cell monolayer (1.13 cm² for 12-wellTranswell®); C_(N) is the nominal concentration of the dosing solutionin 5 μM; C_(rfinal) is the cumulative receiver concentration in 5 μM atthe end of the incubation period; C_(dfinal) is the concentration of thedonor in 5 μM at the end of the incubation period.

REFERENCES

-   1. Qin Z, Hu D, Han S, Hong D P, Fink A L. Role of different regions    of α-synuclein in the assembly of fibrils. Biochemistry. 2007 Nov.    20; 46(46):13322-30.-   2. Cooper A A, Gitler A D, Cashikar A, Haynes C M, Hill K J, Bhullar    B, Liu K, Xu K, Strathearn K E, Liu F, Cao S, Caldwell K A, Caldwell    G A, Marsischky G, Kolodner R D, Labaer J, Rochet J C, Bonini N M,    Lindquist S. A-synuclein blocks ER-Golgi traffic and Rab1 rescues    neuron loss in Parkinson's models. Science. 2006 Jul. 21;    313(5785):324-8.-   3. Siegmund B, Zeitz M. Pralnacasan (Vertex Pharmaceuticals).    IDrugs. 2003 February; 6(2): 154-8.

EQUIVALENTS

The foregoing disclosure is considered to be sufficient to enable oneordinary skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the examples provided, sincethe examples are intended as mere illustrations of one or more aspectsof the invention. Other functionally equivalent embodiments areconsidered within the scope of the invention. Various modifications ofthe invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription. Each of the limitations of the invention can encompassvarious embodiments of the invention. It is therefore anticipated thateach of the limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth orillustrated in the drawing. The invention is capable of otherembodiments and of being practiced or of being carried out in variousways.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including” “comprising” or “having” “containing” “involving” andvariations thereof herein is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

All references, patents and patent applications that are recited in thisapplication are incorporated by reference in their entirety.

What is claimed is:
 1. A method for identifying an ICE inhibitor, themethod comprising steps of: providing a plurality of test compounds;contacting test compounds from the plurality with full-lengthα-synuclein in the presence of ICE; and determining whether one or moreof the test compounds inhibits ICE cleave of the full-lengthα-synuclein.
 2. The method of claim 1, wherein α-synuclein cleavage isdetermined by measuring relative levels of full-length α-synuclein andcleaved α-synuclein, wherein a higher ratio of full-length α-synucleinto cleaved α-synuclein in the presence of the test compound as comparedto the control indicates that the test compound is an ICE inhibitor thatinhibits ICE cleavage of α-synuclein.
 3. The method of claim 2, whereinthe fragment of α-synuclein is about 120 amino acids in length.
 4. Themethod of claim 3, wherein the fragment of α-synuclein is 115 aminoacids in length.
 5. The method of claim 3, wherein the fragment ofα-synuclein is 119 amino acids in length.
 6. The method of claim 3,wherein the fragment of α-synuclein is 121 amino acids in length.
 7. Themethod of claim 2, wherein the fragment of α-synuclein is about 20 aminoacids in length.
 8. An α-synuclein antibody that specifically binds to afull-length α-synuclein but not to ICE-cleaved α-synuclein.
 9. Anα-synuclein antibody that specifically binds to an ICE-cleavedα-synuclein but not to a full-length α-synuclein.
 10. The α-synucleinantibody of claim 8 or 9, wherein the antibody is selected from thegroup consisting of: monoclonal antibodies, polyclonal antibodies, Fabfragments, Fab′ fragments, F(ab′)₂ fragments, Fv fragments, diabodies,single-chain antibody molecules and multispecific antibodies.
 11. Amethod for identifying an α-synuclein-cleaving enzymes, the methodcomprising steps of: providing a plurality of caspase enzymes that arecandidate α-synuclein cleaving enzymes; contacting candidate enzymesfrom the plurality a full-length α-synuclein; and determining whetherone or more of the candidate enzymes cleaves the full-length α-synucleininto fragments.
 12. The method of claim 11, wherein the fragmentscomprises a fragment of about 120 amino acids in length.
 13. A methodfor treating synucleinopathy disease, disorder or condition in apatient, the method comprising: administering to a patient sufferingfrom or susceptible to a synucleinopathy disease, disorder or conditiona composition comprising: an amount of an ICE inhibitor sufficient toinhibit cleavage of α-synuclein by ICE.
 14. The method of claim 13,wherein the synucleinopathy disease, disorder or condition isParkinson's disease, dementia, or multiple system atrophy.
 15. Themethod of claim 14, wherein the Parkinson's disease is anautosomal-dominant Parkinson's disease.
 16. The method of claim 14,wherein the synucleinopathy disease, disorder or condition ischaracterized by the presence of Lewy bodies.